Milking frequency and effect of fill time on the production and conformation of the udder of female donkeys of the Nordestino ecotype

Milking frequency and effect of fill time on the production and conformation of the udder of female donkeys of the Nordestino ecotype | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Milking frequency and effect of fill time on the production and conformation of the udder of female donkeys of the Nordestino ecotype Karoline Cardoso Santana, Jorge Eduardo Cavalcante Lucena, Alycia Kayla da Silva Pinheiro, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8167477/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The present research aimed to assess the effect of milking frequency and the relationship between udder fill time and morphophysiological characteristics of udder and teats, as well as milk production by female donkeys of the Nordestino ecotype. A group of 15 female donkeys between 3 and 7 years old was employed. The study was split into two trials: The first assessed the effects of different udder fill times (2, 3, and 4 hours) on milk production and mammary morphometry, while the second analyzed the impact of milking frequency (once, twice, or three times a day) on milk production and somatic cell count (SCC). Trial 1 showed fill time of 4 h resulted in greater milk volume obtained (313 g), with a significant increase in udder length (4.5%), depth (3.6%), and teat diameter (9.7%) (P < 0.05). Most morphometric variables had a positive correlation with milk production, with the highest coefficients observed for udder and teat length. In trial 2, increasing milking frequency from one to three times a day led to an increase by up to 457% in milk production. SCC did not vary between treatments, which suggests increasing milking frequency did not compromise mammary health. It is concluded that udder fill time of 4 h and milking three times a day favor milk production in donkeys of the Nordestino ecotype. The moderate to strong correlations between morphometric characteristics and milk production suggest potential use of those parameters as criteria in genetic selection programs targeting donkey milk production. somatic cell milk morphometry teats Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Milking frequency and interval are key factors in milk production and directly impact the secreting efficiency of the mammary gland and lactation persistence. Studies have shown more frequent milking, particularly in early lactation, is associated with greater cellular proliferation and differentiation, in addition to reducing apoptosis rates of secreting cells, which contributes to maintaining production over the lactation cycle (Wall and McFadden 2007 ). However, intensifying milking must be assessed carefully since too short intervals between sessions may compromise the recovery of the teat sphincter, thus increasing the risk of infections such as mastitis (Carneiro 2009). Udder fill time is another critical aspect since it regulates milk accumulation between milkings and impacts secretion dynamics. Longer intervals favor the storage of larger volumes, while short periods may limit the functional recovery of teats and negatively affect mammary health (Svennersten-Sjaunja and Olsson 2005 ). D’Alessandro et al. ( 2015 ) pointed out that, in female donkeys, udder fill time, especially in 3 h intervals, may impact both the amount and the physicochemical quality of milk. In addition to physiological and functional aspects, morphological characteristics of the udder and teats also directly influence milk production and quality. Conformation, size and symmetry of the mammary gland are associated with milking efficiency and secreted volume (Yu et al. 2021 ). In dairy cows, for example, a positive correlation is observed between udder circumference and production capacity (Gašparík et al. 2023 ). In the case of donkeys, the location and anatomy of the udder and teats play an additional role in preserving the microbiological quality of milk. Higher or more protected mammary glands tend to reduce the risk of contact with contaminating agents in the environment, which contributes to maintaining milk purity. Therefore, the antimicrobial efficacy of donkey milk is not only thanks to the presence of bioactive compounds such as lactoferrin, lysozyme, immunoglobulins, and fatty acids, but also to anatomical factors that favor the protection and integrity of milk secretion (Khan et al. 2024 ). Therefore, the present research aimed to assess the effect of milking frequency and the relationship between udder fill time, morphophysiological characteristics of udder and teats, and milk production by female donkeys of the Nordestino ecotype. 2. Material and Methods 2.1. Animals The study used 15 female Nordestino donkeys ( Equus asinus ) between 3 and 7 years old with mean live weight of 185 ± 30 kg. Prior to the experimental phase, the animals underwent a deworming protocol with ivermectin and pyrantel, were given booster vaccine shots against tetanus and rabies, and were submitted to nutritional adjustments for uniform body score. 2.2. Nutritional management The 15 females remained in pens with native pasture throughout the gestational period, with water and mineral lick ad libitum , and their body score was monitored monthly. At 11 months of gestation, the animals were transferred to the maternity pen. During the experimental period (postpartum), they were fed 2.0% of their live weight in dry matter, at 60% Tifton 85 ( Cynodon dactylon ) hay and 40% concentrate feed (Table 1 ) provided twice a day at 5 a.m. and 3 p.m., in addition to free access to water and mineral lick. Table 1 Bromatological composition of the diet. Composition (g/kg DM) Tifton 85 Concentrate Dry matter 809.87 890.17 Ash 71.80 85.70 Digestible energy (Mcal) - 3330.00 Crude protein 62.60 200.00 Ether extract 15.08 - Calcium - 12.00 Phosphorus - 6.00 NDF 1 827.70 - ADF 2 385.40 - 1 Neutral detergent fiber. 2 Acid detergent fiber The study began on the 15th day postpartum for each female donkey and lasted for 120 days, split into two consecutive trials of 60 days each. 2.3. Experiment 1 – Relationship between udder fill time, morphological characteristics, and milk production 2.3.1. Experimental design Trial 1 was conducted between the 15th and 75th days of lactation following a randomized block design in which each female represented one block. The treatments consisted of different times during which mother and offspring were kept separate for the udder to fill (2, 3, and 4 h) with 20 days for each interval. Separating the mothers and offspring at 5 a.m., when they were fed the diet, was also used as reference to apply the udder filling intervals prior to milking (Fig. 1 ). Hence, in the 2 h interval, the mothers were milked at 7 a.m.; in the 3 h interval, at 8 a.m., and in the 4 h interval, at 9 a.m. 2.3.2. Measuring udder and teats Immediately before milking, the udder and teats were measured with a measuring tape following the protocol described by Chirgin et al. ( 2016 ) with adaptations (Figs. 2 and 3 ): Teat diameter – measuring tape around the right and left teats; Udder height from the ground – distance between the udder floor and the ground; Distance between teats – distance between the extremities of right and left teats; Udder circumference – measuring tape around the udder; Udder depth – distance from the base of the teat to the abdominal wall; Udder width – distance between the external side spots of the udder along the line going through the anterior edge of the teats; Teat length – distance between the base and extremity of the teats; Udder length – distance between the anterior and posterior udder edge. 2.3.3. Milking management The animals were milked daily at the holding pens in a roofed area with rubber floor. It was performed once a day at the time defined for each treatment (2, 3, and 4 h) without the foal present. Before each milking, utensils, the hands of the milker, and donkey teats were hygienized with water to remove more evident dirt followed by drying with paper towels, immersion of teats in a 0.5% iodine solution for 30 seconds, and again drying with paper towels. The first three milk squirts were used to check for alterations in the milk, such as clumps, pus, blood, or abnormal coloration, which could indicate clinical mastitis. The animals were milked manually by manipulating one teat at a time until milk ceased coming out. The samples collected were weighed on a Toledo® commercial digital scale with 0.001 kg precision. 2.3.4. Statistical analyses Milk production data and morphometric measurements were submitted to normality test. Next, they were submitted to analysis of variance and the means were compared by Tukey’s test (P < 0.05). They were also submitted to Spearman’s correlation analysis at 5% significance. 2.4. Experiment 2 – Effect of milking frequency on milk production and SCC 2.4.1. Experimental design Trial 2 was carried out between the 76th and 135th days of lactation using a completely randomized design with three treatments: one, two, or three daily milkings with five animals per group. 2.4.2. Milking management The females assigned to one daily milking were milked at 8:30 a.m., the ones in the group of two daily milkings were milked at 8:30 a.m. and 12:30 p.m., while those in the group of three daily milkings were milked at 8:30 a.m., 12:30 p.m., and 4:30 p.m. In all treatments, the mothers were separated from their offspring for 3 h for the udder to fill. The female donkeys were manually milked while the foals were not present always at the same place and adopting the same safety and hygiene measures described for trial 1, with the samples being weighed by the end of the process. 2.4.3. Somatic cell count The samples used for somatic cell count (SCC) were collected in the first and last days of the experimental period, corresponding to the 76th and 135th days of lactation, respectively. The milk samples were stored in properly identified 40 mL sterile plastic flasks containing bronopol preservative that were refrigerated and sent to the Milk Clinic laboratory at the School of Agriculture Luis de Queiroz ( Escola Superior de Agricultura Luiz de Queiroz - Esalq) of the University of São Paulo. At the laboratory, the samples were submitted to flow cytometry analysis to quantify somatic cells present in the raw milk. The SCC results were expressed in number of cells per mL of milk and used to indicate the occurrence of inflammatory processes such as subclinical mastitis over the experimental period. 2.4.4. Statistical analyses The data were submitted to normality test. The milk production data did not exhibit normal distribution and were submitted to non-parametric statistical analysis. In order to compare total milk production among the experimental groups, the data were submitted to Kruskal-Wallis test and the medians were compared by Dunn’s test. On the other hand, SCC data, both before and after 60 days of milking, showed normal distribution when submitted to paired T-test. 3. Results Extending udder fill time from 2 to 4 h resulted in a greater volume of milk obtained and increased udder and teat dimensions (P < 0.05) (Table 2 ). After 4 h separated from the foals, milking the female donkeys achieved 313 g of milk per animal on average. That corresponds to an increase by 40% in relation to the average 188 g of milk obtained when the mothers remained only 2 h separated from the offspring. Table 2 Daily milk production (g) and morphometric measurements (cm) of the udder and teats of female donkeys of the Nordestino ecotype submitted to three different udder fill times and their respective coefficients of variation (CV). Variables Udder fill time (hours) CV (%) 2 3 4 Milk production (g) 188.76 c 249.34 b 312.38 a 42.24 Udder length (cm) 25.80 b 26.75 a 27.01 a 5.42 Udder width (cm) 26.05 c 26.43 b 26.74 a 5.13 Udder circumference (cm) 56.05 b 56.88 a 57.19 a 4.15 Udder depth (cm) 7.55 c 8.17 a 7.83 b 9.33 Udder height from the ground (cm) 66.65 a 66.50 ab 66.25 b 2.03 Right teat length (cm) 3.62 c 4.08 a 3.89 b 12.18 Left teat length (cm) 3.68 b 3.94 a 3.96 a 13.40 Right teat diameter (cm) 6.44 b 6.55 a 7.19 a 10.57 Left teat diameter (cm) 6.41 b 6.48 b 7.04 a 9.15 Distance between teats (cm) 6.62 c 6.91 b 7.05 a 7.76 Different letters in the rows indicate difference between udder fill times by Tukey’s test (P < 0.05). With that extension of udder fill time, udder length was the measurement with the highest percent increase (4.5%). Udder width, circumference, and depth showed increases by 2.6%, 2.0%, and 3.6%, respectively, while udder height from the ground was the only measurement that decreased with a reduction by 0.6% Regarding teat morphometry, extending udder fill time from 2 h to 4 h had the greatest effect on teat diameter, with an increase by 9.7% (taking into account both the right and left teats). Teat length and distance between teats increased by 7.0% and 6.0%, respectively. Regardless of udder fill time, a significant correlation was observed between milk production and most udder and teat morphometric measurements (Table 3 ). Among the measurements assessed, udder width was not significantly associated with any other characteristic. Moreover, the relationship between milk production and udder height from the ground was significant only for the fill time of 2 h. In contrast, all teat morphometric measurements were correlated with milk production irrespective of the treatment adopted. Table 3 Correlation between milk production and morphometric measurements of the udder and teats of female donkeys of the Nordestino ecotype. Udder measurements 2 hours 3 hours 4 hours P-value r P-value r P-value r Length < 0.0001 0.5317 < 0.0001 0.4062 < 0.0001 0.4483 Width 0.6888 - 0.0865 - 0.0824 - Circumference 0.0021 0.1825 0.0054 0.1662 0.5000 - Depth 0.0004 0.2116 0.3009 - < 0.0001 0.3008 Udder height from the ground 0.0049 0.1673 0.7413 - 0.3157 - Teat measurements Right teat length < 0.0001 0.5637 < 0.0001 0.3809 < 0.0001 0.4408 Left teat length < 0.0001 0.6425 < 0.0001 0.5078 < 0.0001 0.4900 Right teat diameter < 0.0001 0.4093 < 0.0001 0.2730 < 0.0001 0.4168 Left teat diameter < 0.0001 0.3870 < 0.0001 0.2730 < 0.0001 0.4253 Distance between teats < 0.0001 0.2329 0.0233 0.1358 < 0.0001 0.3772 Although most morphometric measurements were correlated with milk production, moderate to high Spearman’s correlation coefficients (Field 2013 ) were observed only between milk production and udder length (Fig. 4 ), teat length (Fig. 5 ), and teat diameter. A 457% increase in milk production was found when going from one daily milking (131 g of milk) to three daily milkings (724 g of milk) (Table 4 ). Table 4 Median and lower and upper limits of milk production (g) of female donkeys of the Nordestino ecotype submitted to one, two, or three daily milkings. Treatments Milking time 8:30 a.m. 12:30 p.m. 4:30 p.m. One milking 0.131 (0.013–0.435) - - Two milkings 0.126 (0.019–0.420) 0.119 (0.013–0.523) - Three milkings 0.146 (0.030–0.757) 0.281 (0.019–0.614) 0.298 (0.049–0.627) Total 0.131 c (0.013–0.435) 0.250 b (0.064–0.735) 0.724 a (0.250–1.377) Medians followed by different letters in the row indicate difference in milk production according to Dunn’s test (P < 0.0001). Concerning the relationship between milking frequency and SCC, no difference (P = 0.6657) was found between SCC values in the samples collected before and after 60 days of milking in any treatment assessed (one, two, or three daily milkings) (Table 5 ). Table 5 Average somatic cell count (×10 3 cells/mL) in milk samples collected before and after 60 days of milking from female donkeys of the Nordestino ecotype submitted to one, two, or three daily milkings. One milking Two milkings Three milkings Before 6.25 ± 1.26 3.50 ± 0.58 7.33 ± 4.51 After 5.75 ± 3.59 5.25 ± 1.71 4.00 ± 1.00 4. Discussion 4.1. Udder fill time The alveolar tissue of the udder is responsible for milk secretion, with is later stored in the teat and udder cisterns, as well as in the alveolar ducts and lumens. When those structures reach maximum storage capacity, the secretion process is interrupted (Canisso et al. 2021 ). In the present study, the interval of 4 h for udder fill, in addition to providing a longer period for milk synthesis, allowed for better use of udder storage capacity of the female donkeys when compared with the intervals of 2 and 3 h (Table 2 ). Unlike ruminants, healthy foals in their first week of life suckle up to seven times an hour, with each suckling lasting between 1 and 2 min. In the fourth week of life, that frequency decreases to approximately three times per hour (Carson and Wood-Gush 1983 ). Unlike bovine calves, which often use artificial milk feeding without the presence of the mother (Filho et al. 2018 ), foals are more dependent on their mothers for feeding and care (French 1998 ). In the present study, the foals started the experiment at only two weeks of age. Therefore, adopting intervals above 4 h for udder fill might compromise the health, development, and well-being of the offspring. That concern prevented extending the time the mother and offspring were kept separated and determining the interval required for the udder to reach its maximum milk storage capacity. 4.2. Milk production When the mean milk production of female donkeys of the Nordestino ecotype, obtained with 4 h interval for udder fill, is related to the mean body weight of those animals (expressed as L/100 kg live weight), milk production was higher than the values for the Jiangyue (Guo et al. 2007 ), Pêga (Santos et al. 2023 ), Narsi and North African (Aroua et al. 2024), and Dezhou (Zhou et al. 2025 ) breeds reported in studies that also performed manual milking (Table 6 ). Table 6 Body weight, estimated milk production values in a single milking, and milk production per 100 kg live weight of female donkeys in the present study and in works on different donkey breeds. Reference Breed/ecotype Body weight (kg) Milk production (g) Milk production (g) per 100 kg of body weight Present study Nordestino 185.00 313.00 169.19 Guo et al. ( 2007 ) Jiangyue 210.00 320.00 152.38 Santos et al. ( 2023 ) Pêga 242.00 305.00 126.03 Aroua et al. (2024) Narsi and North African 192.00 465.00 242.19 Zhou et al. ( 2025 ) Dezhou 284.00 428.92 151.03 Unlike donkey breeds that have been selected for that purpose for a long time, such as the European breeds Ragusana and Martina Franca, there has never been genetic improvement programs targeting the selection of females of the Nordestino ecotype for milk production. Those animals were submitted to natural selection and developed in the Brazilian Semi-Arid, a region characterized by feed of low nutritional value for most of the year (Carneiro et al. 2018 ). In this sense, providing a more complete and balanced diet made up of 60% hay and 40% concentrate, likely more nutritive than the feed available in the Caatinga biome, was sufficient for the females to have higher milk production than three of the four breeds compared in Table 6 . That result, allied with the lack of specific genetic selection for milk production, shows the potential of the Nordestino donkey for dairy farming as long as carried out in a professional and technified manner. To that end, it is essential to implement a genetic selection program targeting that purpose, in addition to adopting proper practices for nutritional, sanitary, and milking management. When compared with horses, the milk production of the Nordestino ecotype also stands out. Dias et al. ( 2025 ) determined the total milk production of female Nordestino donkeys by weighing foals before and after suckling and found average production of 5.0 kg of milk per day. When the total milk production is related to the weight of the female donkeys, those authors found an average of 2.70 kg of milk/100 kg live weight. That result is higher than the one found by Santos et al. ( 2005 ), who used the same methodology of weighing foals to estimate total milk production capacity of Mangalarga Marchador mares and reported an average of 2.06 kg of milk/100 kg live weight. In other words, the Nordestino ecotype, with mean weight of 185 kg, has higher total milk production in relation to weight than Mangalarga Marchador mares, which weighed between 415 and 440 kg. According to Santos and Zanine ( 2006 ), the lack of the continued stimulus provided by the presence of the foal during milking has a significant impact on milk production since regular contact between the mother and offspring is essential to stimulate milk synthesis and release. In this context, the result by Dias et al. ( 2025 ), as well as the observation in the present study, support that idea. In the work by Dias et al. ( 2025 ), the female donkeys produced 5.0 kg of milk in 24 h, the equivalent of 416.66 g every 2 h. Thus, in a 4 h interval, the production reported by Dias et al. ( 2025 ) was 833.33 g of milk, which is 62.4% higher than what was found in the present study (313.00 g). The discrepancy between values is likely due to the method employed to obtain the milk: Whereas in the study by Dias et al. ( 2025 ) milk extraction was done directly by the foal, the present study employed manual milking without the foal present. Additionally, the present study observed that few minutes after milking and reuniting the mothers and offspring, udder volume increased again. The milk was likely removed from the cisterns and teats, however, since donkey milk is alveolar, manual milking did not completely remove residual milk. It is worth pointing out that, despite the limited productive capacity of the species, the interest and investment in the selection of the Nordestino ecotype for milk production are justified by the therapeutic benefits of donkey milk and by its classification as a medicinal food, which gives it high added value. Furthermore, the Semi-Arid in the Northeast region of Brazil has low Human Development Index (about 0.550) (Pnud 2013) and exploiting donkey milk in the region may significantly contribute to increasing the income of rural producers. 4.3. Morphological characteristics of the udder and teats Knowing the dimensions of the udder of female donkeys may contribute to the selection of more productive animals since, in addition to genetics, the area taken up by alveoli and ducts, as well as the size of the cistern, directly impact milk production and storage capacity (Ayadi et al. 2003 ). The increase in udder measurements observed between the fill times of 2 and 4 h (Table 3 ) was expected since the longer interval allows for greater milk secretion and accumulation, which raises intramammary pressure and, consequently, alters udder conformation. Among the measurements assessed, the greatest variation was found in udder length, which can be attributed to the bidimensional nature of the determination. That measurement took into account the distance between the anterior and posterior edges of the udder, going around its base ventrally in the cranial-caudal direction. Therefore, in addition to length itself, the measurement also reflected udder depth, comprising not only the area corresponding to the cistern, but also regions with high concentration of alveolar tissue and ducts, directly influenced by the volume of milk stored. The increase in udder fill time from 2 to 4 h significantly impacted teat morphometry, showing structural changes in response to milk accumulation. Teat diameter was the parameter most affected by the change in milking interval, which suggests greater tissue distension with the longer fill time, possibly associated with increased intramammary pressure. It is worth pointing out that, in donkeys, each teat has two cisterns, which may result in a larger storage volume (De Palo et al. 2022 ) and contribute to greater milk storage capacity in each teat. According to the studies by Chirgin et al. ( 2016 ) and Salari et al. ( 2023 ), larger animals tend to have teats with greater dimensions, characterized by greater length, diameter, and distance between the extremities. Those morphological parameters directly influence milk storage capacity and milking efficiency. However, few studies have investigated the relationship between udder fill time and teat morphology, particularly in donkeys. Understanding those morphological variations enables enhancing reproductive and productive management strategies and contributes to the selection of breeds with more appropriate anatomic characteristics for milk production. 4.4. Correlation between morphometric characteristics and milk production The results regarding the influence of morphometric characteristics of the udder and teats (Table 3 ) show a positive association with milk production. Among the variables assessed, udder length showed a moderate correlation with all milking times (Fig. 4 ), which suggests that measurement may indirectly reflect the amount of secretory tissue present in the mammary gland (Davis et al., 2016 ). That result suggests female donkeys with longer udders have greater productive capacity, possibly for having a larger functional area for milk synthesis and storage. Likewise, teat length and diameter on both the right and left sides showed significant correlations with milk production, with the highest coefficients observed for left teat length (Table 3 ). Teat morphological characteristics, such as length, may influence the efficiency of the neuroendocrine reflex associated with milk ejection. Since tactile stimulation is the main trigger for oxytocin, longer teats would tend to provide a greater area of contact during milking, possibly amplifying the sensory response and, therefore, potentiating oxytocin release. Such effect would be especially relevant in donkeys, whose milk letdown is known to be quick and limited, which requires effective and immediate stimulation to ensure complete milk ejection (Farias et al. 2021 ). Moreover, a strong correlation was observed between left teat length and milk production response (Fig. 5 ). That effect may be associated with the management adopted during milking since the animals were milked manually always from the left side and by the same person. Consequently, it is likely the left teat received more intense and frequent tactile stimulus due to the body placement of the milker and the force applied in a more dominant manner on that side. That stimulus may have caused a slight structural elongation over time through tissue adaptations such as reorganization of conjunctive fibers and increase of the functional length of the teat, a process compatible with the remodeling of soft tissues submitted to continuous traction (Mackey et al. 2008 ). That kind of morphologic asymmetry is common in manual milking, in which the stimulus is not standardized, unlike in mechanical milking systems, where the flow of negative pressure exerted by the teat cups is uniform across all teats, thus minimizing variations between sides (Correa and Host 2020). 4.5. Milking frequency Increasing milking frequency from one to three times a day led to an increase by up to 457% in milk production. That finding is in line with other studies that also assessed the effect of milking frequency in donkeys. D’Alessandro et al. (2012), when studying female donkeys of the Martina Franca breed, observed that three daily milkings resulted in greater milk productions when compared with a single milking, with no compromise to mammary health. However, excessive milkings (six times a day) did not lead to additional gains and increased the SCC, which suggests a physiological limit for mammary stimulus. Likewise, Alabiso et al. ( 2009 ) reported that increasing daily milking frequency from three to up to eight was associated with increases in milk production and changes in composition, especially fat content. Those authors point out that milk production and fat percentage showed a positive correlation with the number of milkings per day, which suggests frequent mammary stimulus favors the secreting activity of the gland. Those results support milking frequency as a key factor in modulating milk production in equidae and that increasing the number of milkings may be explored as a management strategy to maximize yield as long as the physiological limits of the species are respected. In the present study, SCC did not vary significantly between treatments, which suggests increasing milking frequency did not compromise mammary health. Such result is relevant as it suggests that, under proper management conditions, milking can be intensified with no harm to milk quality or animal well-being. D’Alessandro et al. (2012) also reported stability in mammary health indicators of female donkeys submitted to three daily milkings, thus supporting the safety of that protocol. Finally, although the practice of a third milking is traditionally associated with high-production bovine herds (Lima et al. 2011 ), the results obtained with female donkeys in the present study suggest the strategy may be promising also for equidae, as long as it is adapted to their physiological and productive particularities. It is worth noting that, despite the production gains associated with increasing milking frequency, that decision must also take into account economic and structural aspects of the property. Input, energy, labor, and feeding costs may compromise profitability, particularly in low-scale systems or with animals of lower productive potential (Barbosa et al. 2013 ). As pointed out by Teixeira Júnior et al. ( 2017 ), intensifying management is only viable when it is followed by strategies that optimize resources and ensure the efficiency of the other production sectors. Therefore, adopting three daily milkings must be evaluated on a case-by-case basis to seek a balance between productivity, sustainability, and economic viability. It was concluded that udder fill time of 4 h and milking three times a day favor milk production in female donkeys of the Nordestino ecotype. The moderate to strong correlations between morphometric characteristics and milk production suggest potential use of those parameters as criteria in genetic selection programs targeting donkey milk production. Declarations Ethics statement All experimental procedures were carried out in accordance with the guidelines by the Committee of Ethics in Animal Use of the Federal Rural University of Pernambuco (CEUA No. 4521100724). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Universidade Federal Rural de Pernambuco (UFRPE) and the Programa de Pós-Graduação em Ciência Animal e Pastagens (PPGCAP) of Federal University of Pernambuco Agreste (UFAPE). Acknowledgments The authors thank the Universidade Federal Rural de Pernambuco (UFRPE); Programa de Pós-Graduação em Ciência Animal e Pastagens (PPGCAP) of Federal University of Pernambuco Agreste (UFAPE) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Data availability None of the data and models are deposited in any official repository. All data is available on request to corresponding author. References Alabiso M, et al. (2009) The effects of different milking intervals and milking times per day in jennet milk production. Animals 3:543–547. 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Lucena","email":"","orcid":"","institution":"Universidade Federal do Agreste de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"Eduardo Cavalcante","lastName":"Lucena","suffix":""},{"id":556555117,"identity":"fd54f9d6-086e-4741-84b2-68f301e71d3b","order_by":2,"name":"Alycia Kayla da Silva Pinheiro","email":"","orcid":"","institution":"Universidade Federal do Agreste de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Alycia","middleName":"Kayla da Silva","lastName":"Pinheiro","suffix":""},{"id":556555118,"identity":"b8c7eaf3-2e90-4d11-87e2-8e617a0064a7","order_by":3,"name":"Damilly de Souza Araujo Pinto","email":"","orcid":"","institution":"Universidade Federal Rural do Agreste de Pernambuco","correspondingAuthor":false,"prefix":"","firstName":"Damilly","middleName":"de Souza Araujo","lastName":"Pinto","suffix":""},{"id":556555119,"identity":"acdb39a5-6ff0-4f2d-999f-ca7f7b0b04fa","order_by":4,"name":"Igor Masterson de 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4","display":"","copyAsset":false,"role":"figure","size":67663,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8167477/v1/e640fc7e5bccd4d4f16cf0ca.jpg"},{"id":97916837,"identity":"fe4af43c-8309-402c-877a-3c9a27ece340","added_by":"auto","created_at":"2025-12-10 17:43:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76899,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8167477/v1/879f4fb09ab08f3ab53822d6.jpg"},{"id":98774971,"identity":"49086927-3398-427b-95ce-557e44898bf4","added_by":"auto","created_at":"2025-12-22 12:17:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1460046,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8167477/v1/a894c488-03fa-41b3-a203-96c1524962bb.pdf"}],"financialInterests":"","formattedTitle":"Milking frequency and effect of fill time on the production and conformation of the udder of female donkeys of the Nordestino ecotype","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMilking frequency and interval are key factors in milk production and directly impact the secreting efficiency of the mammary gland and lactation persistence. Studies have shown more frequent milking, particularly in early lactation, is associated with greater cellular proliferation and differentiation, in addition to reducing apoptosis rates of secreting cells, which contributes to maintaining production over the lactation cycle (Wall and McFadden \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, intensifying milking must be assessed carefully since too short intervals between sessions may compromise the recovery of the teat sphincter, thus increasing the risk of infections such as mastitis (Carneiro 2009).\u003c/p\u003e\u003cp\u003eUdder fill time is another critical aspect since it regulates milk accumulation between milkings and impacts secretion dynamics. Longer intervals favor the storage of larger volumes, while short periods may limit the functional recovery of teats and negatively affect mammary health (Svennersten-Sjaunja and Olsson \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). D\u0026rsquo;Alessandro et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) pointed out that, in female donkeys, udder fill time, especially in 3 h intervals, may impact both the amount and the physicochemical quality of milk.\u003c/p\u003e\u003cp\u003eIn addition to physiological and functional aspects, morphological characteristics of the udder and teats also directly influence milk production and quality. Conformation, size and symmetry of the mammary gland are associated with milking efficiency and secreted volume (Yu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In dairy cows, for example, a positive correlation is observed between udder circumference and production capacity (Gašpar\u0026iacute;k et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the case of donkeys, the location and anatomy of the udder and teats play an additional role in preserving the microbiological quality of milk. Higher or more protected mammary glands tend to reduce the risk of contact with contaminating agents in the environment, which contributes to maintaining milk purity. Therefore, the antimicrobial efficacy of donkey milk is not only thanks to the presence of bioactive compounds such as lactoferrin, lysozyme, immunoglobulins, and fatty acids, but also to anatomical factors that favor the protection and integrity of milk secretion (Khan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, the present research aimed to assess the effect of milking frequency and the relationship between udder fill time, morphophysiological characteristics of udder and teats, and milk production by female donkeys of the Nordestino ecotype.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals\u003c/h2\u003e\u003cp\u003eThe study used 15 female Nordestino donkeys (\u003cem\u003eEquus asinus\u003c/em\u003e) between 3 and 7 years old with mean live weight of 185\u0026thinsp;\u0026plusmn;\u0026thinsp;30 kg. Prior to the experimental phase, the animals underwent a deworming protocol with ivermectin and pyrantel, were given booster vaccine shots against tetanus and rabies, and were submitted to nutritional adjustments for uniform body score.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Nutritional management\u003c/h2\u003e\u003cp\u003eThe 15 females remained in pens with native pasture throughout the gestational period, with water and mineral lick \u003cem\u003ead libitum\u003c/em\u003e, and their body score was monitored monthly. At 11 months of gestation, the animals were transferred to the maternity pen. During the experimental period (postpartum), they were fed 2.0% of their live weight in dry matter, at 60% Tifton 85 (\u003cem\u003eCynodon dactylon\u003c/em\u003e) hay and 40% concentrate feed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) provided twice a day at 5 a.m. and 3 p.m., in addition to free access to water and mineral lick.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBromatological composition of the diet.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComposition (g/kg DM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTifton 85\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentrate\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDry matter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e809.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e890.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e71.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.70\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDigestible energy (Mcal)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3330.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e200.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEther extract\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhosphorus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDF\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e827.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADF\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e385.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e1\u003c/sup\u003e Neutral detergent fiber.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e2\u003c/sup\u003e Acid detergent fiber\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe study began on the 15th day postpartum for each female donkey and lasted for 120 days, split into two consecutive trials of 60 days each.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experiment 1 \u0026ndash; Relationship between udder fill time, morphological characteristics, and milk production\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Experimental design\u003c/h2\u003e\u003cp\u003eTrial 1 was conducted between the 15th and 75th days of lactation following a randomized block design in which each female represented one block. The treatments consisted of different times during which mother and offspring were kept separate for the udder to fill (2, 3, and 4 h) with 20 days for each interval.\u003c/p\u003e\u003cp\u003eSeparating the mothers and offspring at 5 a.m., when they were fed the diet, was also used as reference to apply the udder filling intervals prior to milking (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Hence, in the 2 h interval, the mothers were milked at 7 a.m.; in the 3 h interval, at 8 a.m., and in the 4 h interval, at 9 a.m.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Measuring udder and teats\u003c/h2\u003e\u003cp\u003eImmediately before milking, the udder and teats were measured with a measuring tape following the protocol described by Chirgin et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) with adaptations (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e):\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTeat diameter \u0026ndash; measuring tape around the right and left teats;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUdder height from the ground \u0026ndash; distance between the udder floor and the ground;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eDistance between teats \u0026ndash; distance between the extremities of right and left teats;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUdder circumference \u0026ndash; measuring tape around the udder;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUdder depth \u0026ndash; distance from the base of the teat to the abdominal wall;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUdder width \u0026ndash; distance between the external side spots of the udder along the line going through the anterior edge of the teats;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTeat length \u0026ndash; distance between the base and extremity of the teats;\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUdder length \u0026ndash; distance between the anterior and posterior udder edge.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3. Milking management\u003c/h2\u003e\u003cp\u003eThe animals were milked daily at the holding pens in a roofed area with rubber floor. It was performed once a day at the time defined for each treatment (2, 3, and 4 h) without the foal present. Before each milking, utensils, the hands of the milker, and donkey teats were hygienized with water to remove more evident dirt followed by drying with paper towels, immersion of teats in a 0.5% iodine solution for 30 seconds, and again drying with paper towels.\u003c/p\u003e\u003cp\u003eThe first three milk squirts were used to check for alterations in the milk, such as clumps, pus, blood, or abnormal coloration, which could indicate clinical mastitis. The animals were milked manually by manipulating one teat at a time until milk ceased coming out. The samples collected were weighed on a Toledo\u0026reg; commercial digital scale with 0.001 kg precision.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.4. Statistical analyses\u003c/h2\u003e\u003cp\u003eMilk production data and morphometric measurements were submitted to normality test. Next, they were submitted to analysis of variance and the means were compared by Tukey\u0026rsquo;s test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). They were also submitted to Spearman\u0026rsquo;s correlation analysis at 5% significance.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Experiment 2 \u0026ndash; Effect of milking frequency on milk production and SCC\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Experimental design\u003c/h2\u003e\u003cp\u003eTrial 2 was carried out between the 76th and 135th days of lactation using a completely randomized design with three treatments: one, two, or three daily milkings with five animals per group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Milking management\u003c/h2\u003e\u003cp\u003eThe females assigned to one daily milking were milked at 8:30 a.m., the ones in the group of two daily milkings were milked at 8:30 a.m. and 12:30 p.m., while those in the group of three daily milkings were milked at 8:30 a.m., 12:30 p.m., and 4:30 p.m.\u003c/p\u003e\u003cp\u003eIn all treatments, the mothers were separated from their offspring for 3 h for the udder to fill. The female donkeys were manually milked while the foals were not present always at the same place and adopting the same safety and hygiene measures described for trial 1, with the samples being weighed by the end of the process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. Somatic cell count\u003c/h2\u003e\u003cp\u003eThe samples used for somatic cell count (SCC) were collected in the first and last days of the experimental period, corresponding to the 76th and 135th days of lactation, respectively. The milk samples were stored in properly identified 40 mL sterile plastic flasks containing bronopol preservative that were refrigerated and sent to the Milk Clinic laboratory at the School of Agriculture Luis de Queiroz (\u003cem\u003eEscola Superior de Agricultura Luiz de Queiroz\u003c/em\u003e - Esalq) of the University of S\u0026atilde;o Paulo.\u003c/p\u003e\u003cp\u003eAt the laboratory, the samples were submitted to flow cytometry analysis to quantify somatic cells present in the raw milk. The SCC results were expressed in number of cells per mL of milk and used to indicate the occurrence of inflammatory processes such as subclinical mastitis over the experimental period.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4. Statistical analyses\u003c/h2\u003e\u003cp\u003eThe data were submitted to normality test. The milk production data did not exhibit normal distribution and were submitted to non-parametric statistical analysis. In order to compare total milk production among the experimental groups, the data were submitted to Kruskal-Wallis test and the medians were compared by Dunn\u0026rsquo;s test. On the other hand, SCC data, both before and after 60 days of milking, showed normal distribution when submitted to paired T-test.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eExtending udder fill time from 2 to 4 h resulted in a greater volume of milk obtained and increased udder and teat dimensions (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). After 4 h separated from the foals, milking the female donkeys achieved 313 g of milk per animal on average. That corresponds to an increase by 40% in relation to the average 188 g of milk obtained when the mothers remained only 2 h separated from the offspring.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDaily milk production (g) and morphometric measurements (cm) of the udder and teats of female donkeys of the Nordestino ecotype submitted to three different udder fill times and their respective coefficients of variation (CV).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eVariables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eUdder fill time (hours)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCV (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMilk production (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e188.76\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e249.34\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e312.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e42.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUdder length (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUdder width (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.74\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUdder circumference (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e56.88\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUdder depth (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.55\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUdder height from the ground (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e66.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRight teat length (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.62\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e12.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeft teat length (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.96\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRight teat diameter (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.44\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10.57\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeft teat diameter (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDistance between teats (cm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.62\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent letters in the rows indicate difference between udder fill times by Tukey\u0026rsquo;s test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWith that extension of udder fill time, udder length was the measurement with the highest percent increase (4.5%). Udder width, circumference, and depth showed increases by 2.6%, 2.0%, and 3.6%, respectively, while udder height from the ground was the only measurement that decreased with a reduction by 0.6%\u003c/p\u003e\u003cp\u003eRegarding teat morphometry, extending udder fill time from 2 h to 4 h had the greatest effect on teat diameter, with an increase by 9.7% (taking into account both the right and left teats). Teat length and distance between teats increased by 7.0% and 6.0%, respectively.\u003c/p\u003e\u003cp\u003eRegardless of udder fill time, a significant correlation was observed between milk production and most udder and teat morphometric measurements (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Among the measurements assessed, udder width was not significantly associated with any other characteristic. Moreover, the relationship between milk production and udder height from the ground was significant only for the fill time of 2 h. In contrast, all teat morphometric measurements were correlated with milk production irrespective of the treatment adopted.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelation between milk production and morphometric measurements of the udder and teats of female donkeys of the Nordestino ecotype.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eUdder measurements\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e2 hours\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e3 hours\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e4 hours\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003er\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLength\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5317\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.4062\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4483\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWidth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.6888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0865\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.0824\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCircumference\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1825\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.1662\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.5000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDepth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.3009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.3008\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUdder height from the ground\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.0049\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1673\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.7413\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.3157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTeat measurements\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRight teat length\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5637\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.3809\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4408\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeft teat length\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.6425\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.5078\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4900\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRight teat diameter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.4093\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.2730\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4168\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLeft teat diameter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3870\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.2730\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.4253\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDistance between teats\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.2329\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.0233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.1358\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.3772\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAlthough most morphometric measurements were correlated with milk production, moderate to high Spearman\u0026rsquo;s correlation coefficients (Field \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) were observed only between milk production and udder length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), teat length (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and teat diameter.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA 457% increase in milk production was found when going from one daily milking (131 g of milk) to three daily milkings (724 g of milk) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMedian and lower and upper limits of milk production (g) of female donkeys of the Nordestino ecotype submitted to one, two, or three daily milkings.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTreatments\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMilking time\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8:30 a.m.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12:30 p.m.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4:30 p.m.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOne milking\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.131 (0.013\u0026ndash;0.435)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTwo milkings\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.126 (0.019\u0026ndash;0.420)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.119 (0.013\u0026ndash;0.523)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eThree milkings\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.146 (0.030\u0026ndash;0.757)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.281 (0.019\u0026ndash;0.614)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.298 (0.049\u0026ndash;0.627)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.131\u003csup\u003ec\u003c/sup\u003e (0.013\u0026ndash;0.435)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.250\u003csup\u003eb\u003c/sup\u003e (0.064\u0026ndash;0.735)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.724\u003csup\u003ea\u003c/sup\u003e (0.250\u0026ndash;1.377)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eMedians followed by different letters in the row indicate difference in milk production according to Dunn\u0026rsquo;s test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eConcerning the relationship between milking frequency and SCC, no difference (P\u0026thinsp;=\u0026thinsp;0.6657) was found between SCC values in the samples collected before and after 60 days of milking in any treatment assessed (one, two, or three daily milkings) (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAverage somatic cell count (\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/mL) in milk samples collected before and after 60 days of milking from female donkeys of the Nordestino ecotype submitted to one, two, or three daily milkings.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOne milking\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTwo milkings\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eThree milkings\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBefore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAfter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Udder fill time\u003c/h2\u003e\u003cp\u003eThe alveolar tissue of the udder is responsible for milk secretion, with is later stored in the teat and udder cisterns, as well as in the alveolar ducts and lumens. When those structures reach maximum storage capacity, the secretion process is interrupted (Canisso et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present study, the interval of 4 h for udder fill, in addition to providing a longer period for milk synthesis, allowed for better use of udder storage capacity of the female donkeys when compared with the intervals of 2 and 3 h (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnlike ruminants, healthy foals in their first week of life suckle up to seven times an hour, with each suckling lasting between 1 and 2 min. In the fourth week of life, that frequency decreases to approximately three times per hour (Carson and Wood-Gush \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Unlike bovine calves, which often use artificial milk feeding without the presence of the mother (Filho et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), foals are more dependent on their mothers for feeding and care (French \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In the present study, the foals started the experiment at only two weeks of age. Therefore, adopting intervals above 4 h for udder fill might compromise the health, development, and well-being of the offspring. That concern prevented extending the time the mother and offspring were kept separated and determining the interval required for the udder to reach its maximum milk storage capacity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Milk production\u003c/h2\u003e\u003cp\u003eWhen the mean milk production of female donkeys of the Nordestino ecotype, obtained with 4 h interval for udder fill, is related to the mean body weight of those animals (expressed as L/100 kg live weight), milk production was higher than the values for the Jiangyue (Guo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), P\u0026ecirc;ga (Santos et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Narsi and North African (Aroua et al. 2024), and Dezhou (Zhou et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) breeds reported in studies that also performed manual milking (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBody weight, estimated milk production values in a single milking, and milk production per 100 kg live weight of female donkeys in the present study and in works on different donkey breeds.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBreed/ecotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBody weight (kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMilk production (g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMilk production (g) per 100 kg of body weight\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePresent study\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNordestino\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e185.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e313.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e169.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGuo et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eJiangyue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e210.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e320.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e152.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSantos et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP\u0026ecirc;ga\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e242.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e305.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e126.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAroua et al. (2024)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNarsi and North African\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e192.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e465.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e242.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZhou et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDezhou\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e284.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e428.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e151.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eUnlike donkey breeds that have been selected for that purpose for a long time, such as the European breeds Ragusana and Martina Franca, there has never been genetic improvement programs targeting the selection of females of the Nordestino ecotype for milk production. Those animals were submitted to natural selection and developed in the Brazilian Semi-Arid, a region characterized by feed of low nutritional value for most of the year (Carneiro et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this sense, providing a more complete and balanced diet made up of 60% hay and 40% concentrate, likely more nutritive than the feed available in the Caatinga biome, was sufficient for the females to have higher milk production than three of the four breeds compared in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. That result, allied with the lack of specific genetic selection for milk production, shows the potential of the Nordestino donkey for dairy farming as long as carried out in a professional and technified manner. To that end, it is essential to implement a genetic selection program targeting that purpose, in addition to adopting proper practices for nutritional, sanitary, and milking management.\u003c/p\u003e\u003cp\u003eWhen compared with horses, the milk production of the Nordestino ecotype also stands out. Dias et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) determined the total milk production of female Nordestino donkeys by weighing foals before and after suckling and found average production of 5.0 kg of milk per day. When the total milk production is related to the weight of the female donkeys, those authors found an average of 2.70 kg of milk/100 kg live weight. That result is higher than the one found by Santos et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), who used the same methodology of weighing foals to estimate total milk production capacity of Mangalarga Marchador mares and reported an average of 2.06 kg of milk/100 kg live weight. In other words, the Nordestino ecotype, with mean weight of 185 kg, has higher total milk production in relation to weight than Mangalarga Marchador mares, which weighed between 415 and 440 kg.\u003c/p\u003e\u003cp\u003eAccording to Santos and Zanine (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), the lack of the continued stimulus provided by the presence of the foal during milking has a significant impact on milk production since regular contact between the mother and offspring is essential to stimulate milk synthesis and release. In this context, the result by Dias et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), as well as the observation in the present study, support that idea.\u003c/p\u003e\u003cp\u003eIn the work by Dias et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), the female donkeys produced 5.0 kg of milk in 24 h, the equivalent of 416.66 g every 2 h. Thus, in a 4 h interval, the production reported by Dias et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) was 833.33 g of milk, which is 62.4% higher than what was found in the present study (313.00 g). The discrepancy between values is likely due to the method employed to obtain the milk: Whereas in the study by Dias et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) milk extraction was done directly by the foal, the present study employed manual milking without the foal present. Additionally, the present study observed that few minutes after milking and reuniting the mothers and offspring, udder volume increased again. The milk was likely removed from the cisterns and teats, however, since donkey milk is alveolar, manual milking did not completely remove residual milk.\u003c/p\u003e\u003cp\u003eIt is worth pointing out that, despite the limited productive capacity of the species, the interest and investment in the selection of the Nordestino ecotype for milk production are justified by the therapeutic benefits of donkey milk and by its classification as a medicinal food, which gives it high added value. Furthermore, the Semi-Arid in the Northeast region of Brazil has low Human Development Index (about 0.550) (Pnud 2013) and exploiting donkey milk in the region may significantly contribute to increasing the income of rural producers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Morphological characteristics of the udder and teats\u003c/h2\u003e\u003cp\u003eKnowing the dimensions of the udder of female donkeys may contribute to the selection of more productive animals since, in addition to genetics, the area taken up by alveoli and ducts, as well as the size of the cistern, directly impact milk production and storage capacity (Ayadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe increase in udder measurements observed between the fill times of 2 and 4 h (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) was expected since the longer interval allows for greater milk secretion and accumulation, which raises intramammary pressure and, consequently, alters udder conformation. Among the measurements assessed, the greatest variation was found in udder length, which can be attributed to the bidimensional nature of the determination. That measurement took into account the distance between the anterior and posterior edges of the udder, going around its base ventrally in the cranial-caudal direction. Therefore, in addition to length itself, the measurement also reflected udder depth, comprising not only the area corresponding to the cistern, but also regions with high concentration of alveolar tissue and ducts, directly influenced by the volume of milk stored.\u003c/p\u003e\u003cp\u003eThe increase in udder fill time from 2 to 4 h significantly impacted teat morphometry, showing structural changes in response to milk accumulation. Teat diameter was the parameter most affected by the change in milking interval, which suggests greater tissue distension with the longer fill time, possibly associated with increased intramammary pressure. It is worth pointing out that, in donkeys, each teat has two cisterns, which may result in a larger storage volume (De Palo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and contribute to greater milk storage capacity in each teat.\u003c/p\u003e\u003cp\u003eAccording to the studies by Chirgin et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Salari et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), larger animals tend to have teats with greater dimensions, characterized by greater length, diameter, and distance between the extremities. Those morphological parameters directly influence milk storage capacity and milking efficiency. However, few studies have investigated the relationship between udder fill time and teat morphology, particularly in donkeys. Understanding those morphological variations enables enhancing reproductive and productive management strategies and contributes to the selection of breeds with more appropriate anatomic characteristics for milk production.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Correlation between morphometric characteristics and milk production\u003c/h2\u003e\u003cp\u003eThe results regarding the influence of morphometric characteristics of the udder and teats (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) show a positive association with milk production. Among the variables assessed, udder length showed a moderate correlation with all milking times (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which suggests that measurement may indirectly reflect the amount of secretory tissue present in the mammary gland (Davis et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). That result suggests female donkeys with longer udders have greater productive capacity, possibly for having a larger functional area for milk synthesis and storage.\u003c/p\u003e\u003cp\u003eLikewise, teat length and diameter on both the right and left sides showed significant correlations with milk production, with the highest coefficients observed for left teat length (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTeat morphological characteristics, such as length, may influence the efficiency of the neuroendocrine reflex associated with milk ejection. Since tactile stimulation is the main trigger for oxytocin, longer teats would tend to provide a greater area of contact during milking, possibly amplifying the sensory response and, therefore, potentiating oxytocin release. Such effect would be especially relevant in donkeys, whose milk letdown is known to be quick and limited, which requires effective and immediate stimulation to ensure complete milk ejection (Farias et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, a strong correlation was observed between left teat length and milk production response (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). That effect may be associated with the management adopted during milking since the animals were milked manually always from the left side and by the same person. Consequently, it is likely the left teat received more intense and frequent tactile stimulus due to the body placement of the milker and the force applied in a more dominant manner on that side. That stimulus may have caused a slight structural elongation over time through tissue adaptations such as reorganization of conjunctive fibers and increase of the functional length of the teat, a process compatible with the remodeling of soft tissues submitted to continuous traction (Mackey et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThat kind of morphologic asymmetry is common in manual milking, in which the stimulus is not standardized, unlike in mechanical milking systems, where the flow of negative pressure exerted by the teat cups is uniform across all teats, thus minimizing variations between sides (Correa and Host 2020).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Milking frequency\u003c/h2\u003e\u003cp\u003eIncreasing milking frequency from one to three times a day led to an increase by up to 457% in milk production. That finding is in line with other studies that also assessed the effect of milking frequency in donkeys. D\u0026rsquo;Alessandro et al. (2012), when studying female donkeys of the Martina Franca breed, observed that three daily milkings resulted in greater milk productions when compared with a single milking, with no compromise to mammary health. However, excessive milkings (six times a day) did not lead to additional gains and increased the SCC, which suggests a physiological limit for mammary stimulus.\u003c/p\u003e\u003cp\u003eLikewise, Alabiso et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that increasing daily milking frequency from three to up to eight was associated with increases in milk production and changes in composition, especially fat content. Those authors point out that milk production and fat percentage showed a positive correlation with the number of milkings per day, which suggests frequent mammary stimulus favors the secreting activity of the gland.\u003c/p\u003e\u003cp\u003eThose results support milking frequency as a key factor in modulating milk production in equidae and that increasing the number of milkings may be explored as a management strategy to maximize yield as long as the physiological limits of the species are respected.\u003c/p\u003e\u003cp\u003eIn the present study, SCC did not vary significantly between treatments, which suggests increasing milking frequency did not compromise mammary health. Such result is relevant as it suggests that, under proper management conditions, milking can be intensified with no harm to milk quality or animal well-being. D\u0026rsquo;Alessandro et al. (2012) also reported stability in mammary health indicators of female donkeys submitted to three daily milkings, thus supporting the safety of that protocol.\u003c/p\u003e\u003cp\u003eFinally, although the practice of a third milking is traditionally associated with high-production bovine herds (Lima et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), the results obtained with female donkeys in the present study suggest the strategy may be promising also for equidae, as long as it is adapted to their physiological and productive particularities.\u003c/p\u003e\u003cp\u003eIt is worth noting that, despite the production gains associated with increasing milking frequency, that decision must also take into account economic and structural aspects of the property. Input, energy, labor, and feeding costs may compromise profitability, particularly in low-scale systems or with animals of lower productive potential (Barbosa et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As pointed out by Teixeira J\u0026uacute;nior et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), intensifying management is only viable when it is followed by strategies that optimize resources and ensure the efficiency of the other production sectors. Therefore, adopting three daily milkings must be evaluated on a case-by-case basis to seek a balance between productivity, sustainability, and economic viability.\u003c/p\u003e\u003cp\u003eIt was concluded that udder fill time of 4 h and milking three times a day favor milk production in female donkeys of the Nordestino ecotype. The moderate to strong correlations between morphometric characteristics and milk production suggest potential use of those parameters as criteria in genetic selection programs targeting donkey milk production.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e All experimental procedures were carried out in accordance with the guidelines by the Committee of Ethics in Animal Use of the Federal Rural University of Pernambuco (CEUA No. 4521100724).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Universidade Federal Rural de Pernambuco (UFRPE) and the Programa de P\u0026oacute;s-Gradua\u0026ccedil;\u0026atilde;o em Ci\u0026ecirc;ncia Animal e Pastagens (PPGCAP) of Federal University of Pernambuco Agreste (UFAPE).\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors thank the Universidade Federal Rural de Pernambuco (UFRPE); Programa de P\u0026oacute;s-Gradua\u0026ccedil;\u0026atilde;o em Ci\u0026ecirc;ncia Animal e Pastagens (PPGCAP) of Federal University of Pernambuco Agreste (UFAPE) and the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eNone of the data and models are deposited in any official repository. All data is available on request to corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlabiso M, et al. (2009) The effects of different milking intervals and milking times per day in jennet milk production. \u003cem\u003eAnimals\u003c/em\u003e 3:543\u0026ndash;547.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAyadi M, Gaja G, Such X, et al. (2003) Use of ultrasonography to estimate cistern size and milk storage at different milking intervals in the udder of dairy cows. \u003cem\u003eJ Dairy Res\u003c/em\u003e 70:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarbosa GL, Silva RG, Lima AL, Oliveira ME (2013) Viabilidade econ\u0026ocirc;mica da terceira ordenha em sistemas de produ\u0026ccedil;\u0026atilde;o de leite com ordenhadeira tipo circuito aberto. \u003cem\u003eArq Bras Med Vet Zootec\u003c/em\u003e 65(4):1123\u0026ndash;1130.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCanisso IF, et al. 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(2025) The milk compositions and blood parameters of lactating Dezhou donkeys changes with lactation stages. \u003cem\u003eVet Med Sci\u003c/em\u003e 11(2)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"somatic cell, milk, morphometry, teats","lastPublishedDoi":"10.21203/rs.3.rs-8167477/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8167477/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe present research aimed to assess the effect of milking frequency and the relationship between udder fill time and morphophysiological characteristics of udder and teats, as well as milk production by female donkeys of the Nordestino ecotype. A group of 15 female donkeys between 3 and 7 years old was employed. The study was split into two trials: The first assessed the effects of different udder fill times (2, 3, and 4 hours) on milk production and mammary morphometry, while the second analyzed the impact of milking frequency (once, twice, or three times a day) on milk production and somatic cell count (SCC). Trial 1 showed fill time of 4 h resulted in greater milk volume obtained (313 g), with a significant increase in udder length (4.5%), depth (3.6%), and teat diameter (9.7%) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Most morphometric variables had a positive correlation with milk production, with the highest coefficients observed for udder and teat length. In trial 2, increasing milking frequency from one to three times a day led to an increase by up to 457% in milk production. SCC did not vary between treatments, which suggests increasing milking frequency did not compromise mammary health. It is concluded that udder fill time of 4 h and milking three times a day favor milk production in donkeys of the Nordestino ecotype. The moderate to strong correlations between morphometric characteristics and milk production suggest potential use of those parameters as criteria in genetic selection programs targeting donkey milk production.\u003c/p\u003e","manuscriptTitle":"Milking frequency and effect of fill time on the production and conformation of the udder of female donkeys of the Nordestino ecotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 17:42:59","doi":"10.21203/rs.3.rs-8167477/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6a53816-f17f-4248-b1f1-a6e237ea6a93","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-13T10:17:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 17:42:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8167477","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8167477","identity":"rs-8167477","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}