Interaction of CP and NCP BVDV with bovine sperm cells on apoptosis and oxidative stress in vitro

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The goal of this study was to see how CP and NCP biotypes of BVDV affected on spermatozoa in vitro . Materials and methods BVDV-free frozen sperm cells were counted and centrifuged to separate viable sperms. CP and NCP BVDV with 3 different doses of 10 5 , 10 4 and 10 3 TCID 50/mL were incubated to10 5 sperm/ml for 2 hours. To assess oxidative stress and apoptosis, the luminescence technique was used to quantify ROS, Caspases 7/3 and 9, as well as ATP. They were repeated 3 times. Results The effect of virus biotypes on plasma membrane integrity has led to a significant drop in both 10 5 and 10 3 CP and NCP BVDV concentrations (P ≤ 0.05). The impact of 10 5 dose of CP biotype compared to 10 4 and 10 3 doses of the same biotype was significant (P ≤ 0.05), but no significant difference was observed between the effect of 10 4 and 10 3 doses. Caspase 3/7 produced showed a significant effect (P ≤ 0.05) of both biotypes on sperm cells apoptosis. Caspase 9 produced showed a significant effect (P ≤ 0.05) of the three dosages of CP in addition to the high dose of NCP on apoptosis. ATP produced showed a significant effect (P ≤ 0.05) of 3 dosages. The effect of 10 5 of CP compared to 10 4 doses and 10 4 to 10 3 doses were significant (P ≤ 0.05). Conclusion We concluded that BVDV biotypes could affect the vital aspects, dynamic features and also induction of oxidative stress and apoptosis on bovine spermatozoa cells in vitro . BVDV CP NCP apoptosis Oxidative stress Sperm In-vitro Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Contrary to its name, which refers to diarrhea, the bovine viral diarrhea virus (BVDV) is among the most dangerous viral illnesses in cattle and other ruminants, exhibiting extensive detrimental consequences on the reproductive tract and fertility. This pathogen was first discovered in 1946 and is one of the germs which can cause substantial economic losses to the dairy industry [ 1 , 2 ]. In immune hosts, the illness is subclinical, also 70 to 90% of animals with acute form do not show clinical manifestations [ 3 ]. BVDV is the first member of the Flaviviridae family and is a single-stranded, enveloped RNA virus. The virus is currently divided into four strains: BVDV genotypes 1 and 2, borderline disease virus (BDV), and classical swine fever virus (CSFV). BVDV isolates can be classified into Cytopathic (CP) and Non-Cytopathic (NCP) biotypes based on their disastrous impacts on the cells. CP isolates, unlike NCP, can trigger vacuole formation in the cytoplasm along with cell death in the culture medium [ 4 ]. NCP biotypes are more abundant in nature and comprise 60 to 90% of isolates. Only NCP biotype strains can cause chronic infection. These strains are assumed to be a common source of CP strains because of a certain nucleotide combination in the NS2-3 genomic region [ 5 ]. The BVDV can grow in both male and female gonads, along with all sections of the female reproductive tract. BVDV infection in cattle can be fatal during pregnancy, especially in the pre-trimester stage, which is riskier. Both biotypes of the virus can infect the fetus and easily penetrate the placental barrier [ 6 ]. BVDV's natural host is cattle and the illness is endemic in cattle herds around the world [ 7 – 9 ]. According to serological surveys conducted in various geographic regions, the prevalence of BVDV infection cattle ranges from 40 to 90 percent in individual cattle and 28 to 66 percent in cattle herds, with 0.5 to 2.5 percent of cattle persistently infected (PI) with the virus [ 7 , 10 , 11 ]. BVDV is found in more than 50 to 100 percent of dairy herds in Iran, according to epidemiological studies [ 12 ]. Direct contact between animals is the most typical way for BVDV to spread [ 4 ]. BVDV is shed in various bodily fluids and excretions by infected cattle, including nasal discharge, saliva, sperm, urine, feces, tears, milk, and uterine flushing [ 13 , 14 ]. BVDV can also be spread during rectal examinations and also during natural breeding or artificial insemination (AI) of cattle with infected bulls' sperm [ 15 , 16 ]. BVDV can infect male reproductive organs, and researchers are looking into the effects of virus infection on testicular function and bull fertility in many studies. Infection of sperm with this agent might come from four different places: 1) Permanent infection of male fetus throughout gestation (vertical transmission), 2) Acute infection of the bull following development of immune system, 3) Chronic infection of the testicle after protracted acute infection, and 4) Persistent testicular infection out of an unidentified source [ 17 ]. In addition to, infection of in vivo and in vitro embryos can be accomplished in three ways: 1) donor cows, 2) recipient animals, and 3) animal-based laboratory products such as bovine fetal serum (BFS). Each of these factors has the potential to influence agent transmission. Following both acute and persistent infection, seminal vesicles and the prostate gland can be an ideal refuge for BVDV multiplication and subsequent transfer to the semen of bulls [ 18 ]. A few studies found that neither acutely infected nor PI bulls had any evident changes in sperm quality or semen. Several investigations, on the other hand, found irregularities such as decreased sperm volume, disrupted sperm concentration and motility, and increased sperm malformations [ 19 ]. Cows bred with PI bull's sperm had a 38 percent conception rate compared to 66 percent for those bred with uninfected bull's sperm [ 20 ]. As a result, BVDV infection can affect testicular activity, resulting in spermatozoa anomalies. Semen from these sick bulls could be a cause of infection for sensitive cows, lowering conception rates and fertility in cows after natural breeding or artificial insemination. Because there isn't enough intracytoplasmic space for defense enzymes and antioxidants to concentrate, spermatozoa are extremely sensitive to oxidative damage. Furthermore, the plasma membrane's high amount of unsaturated fatty acids renders it more vulnerable to oxidative stress [ 21 ]. Meanwhile, sperm capacitation generates a lot of reactive oxygen species (ROS), which could also lead to oxidative stress and subsequent functional failure. The cell can be driven to programmed cell death (apoptosis) under these conditions, which is followed by mitochondrial ROS production, depletion of mitochondrial membrane potential, caspase activity, phosphatidylserine overexpression, and DNA oxidative injury [ 22 ]. As a result, any condition that causes an increase in the production of these chemicals in sperm can have an impact on its performance. Disrupting critical elements of the sperm, such as the plasma membrane, mitochondria, or acrosomes, might induce this unfavorable effect. As a result, measuring ROS and apoptotic indicators can reveal how the BVDV mechanism harms bovine spermatozoa [ 21 ]. The goal of this study was to see how CP and NCP BVDV affected the performance of Holstein bull spermatozoa in vitro . Viability, membrane integrity, motility, viral-induced oxidative stress, and apoptosis were all measured in the spermatozoa. Materials and Methods BVDV free sperm samples All utilized sperm samples were checked by PCR before any tests to rule out the existence of the BVD virus [ 24 ]. The BVD virus was not found in any of the sperm sample. Sperm samples BVDV-free bull's frozen sperm which were routinely used in farms for Artificial Insemination (AI) of dairy cows was thawed in a water bath at 37°C and added to the head of a Percoll gradient (45 and 90 percent; Pharmacia, Uppsala, Sweden). The following method was used to distinguish live and dead spermatozoa: the sperm sample was centrifuged for 30 minutes at 2000 g. After centrifugation, the supernatant was removed, and the sperm pellet was re-suspended in TALP + BSA (bovine serum albumin; Sigma-Aldrich, Bornem, Belgium). The sperm pellet was then re-suspended to the desired concentration of 10 5 sperm/ml. frozen sperm samples were taken from a serial number of the national breeding facility for animal production enhancement (Karaj, Iran), which were BVDV-free. Virus High dose (10 5 ), medium dose (10 4 ), and low dose (10 3 ) (TCID50 / ml) CP and NCP biotypes were utilized. The virus was cultivated in a 5 percent fetal calf serum Minimum Essential Medium (MEM) [ 25 ]. Viability, membrane integrity and motility evaluation Viability, membrane integrity and motility were assessed according to the prior experiment performed by the current team [ 24 ]. Bioluminescence Experiment on Sperms The generation and emission of light by a living creature is known as bioluminescence. Luciferase is a frequent enzyme involved in this process. This approach was used to assess the activity of caspase 3 and 7, caspase 9, and ATP in this investigation [ 26 ]. This test was carried out with the aid of an illuminometer. Before beginning the test, the pipes would have been thoroughly cleaned and dried with distilled water and alcohol, resulting in a relative light unit (RLU) of less than 300 [ 27 ]. Preparation of Luciferase The Luciferase enzyme, synthesized in Tarbiat Modares University- Iran, was employed in this study according to the following protocol: The bacteria BL21, which contains this enzyme was used. To be more precise, Firstly, Lactose or IPTG was added to a plasmid expressing Luciferase to expand the expression. Secondly, it was purified on the Ni-NTA nickel sepharose chromatography column. Lastly, one milliliter of elution (protein collected from the column) along with 120 microliters of 60% glycerol was stored in the freezer to be used in later molecular experiments. Cell Lysis In terms of evaluating the activity of Caspase and ATP, the sperm pellet must primarily be lysed. For this purpose, cell culture lysis buffer (CCLR) was applied. The instruction of preparing this buffer is that 10ml glycerol, 231mg potassium dihydrogen phosphate salt (KH2PO4), 37 mg EDTA, 3.18 gr dipotassium mono-hydrogen phosphate, Triton 1%, and 48 µl double Mercaptoethanol was dissolved in deionized distilled water, and the magnet was placed in a tube and stirred and at last made its volume to 100 [ 28 ]. In the presence of ice, 150 µl of CCLR buffer was added to help the sperm pellet lyse. After 50 times of gentle pipetting, they were placed in the shaker section of the refrigerator for 30 minutes to complete dissolution. In the next step, it was centrifuged at 4°C for 10 minutes at 3000 rpm. The obtained solution was evenly distributed in three microtubes for bioluminescence experiments [ 28 ]. The Measurement of Caspase 3 and 7 in Sperms In this experiment, sperm Caspases 3 and 7 against Luciferase enzyme were assessed. These components of the Caspase family play a key role in cellular apoptosis. This method is based on luminogenic Caspase 3 and 7 substrates (containing DEVD tetrapeptide sequence). It implies that following the breakdown of caspases 3 and 7, the luciferase substrate (amino-luciferin) is released, which results in reaction with Luciferase and consequently production/emission of light. In this section, three microliters of sperm lysis were predominantly added to the bioluminescence tubes. Two microliters of luciferase enzyme were added, and at times of zero, five, 10, 15, 20, 25 minutes, the producing bioluminescence was read and recorded in terms of RLU/s by the Illuminator device [ 28 ]. The Measurement of Caspase 9 in Sperms In this section, the activity of Caspase 9 was evaluated. This enzyme plays a crucial role in the innate pathway of apoptosis. This method, which is a homogeneous luminescent test, is based on the luminogenic substrate of caspase 9 (with the LEHD tetrapeptide sequence), which is released as a consequence of the breakdown of caspase 9 by the reaction of luciferase substrate (amino-luciferin) with Luciferase, resulting in light production [ 26 ]. In this part, at first, five microliters of cell lysis were added to the bioluminescence tubes. Two microliters of luciferase enzyme were added to it. Every 30 seconds in 0–10 minutes, the numbers are shown as RLU / s were recorded by the device and the maximum absorbance was reported during this period [ 26 ]. The Measurement of ATP ATP measurement would be another factor to determine sperm cell damage. This method was used for very low concentrations of ATP, which is based on the reaction of ATP with Luciferase and the oxidation of the luciferin catalyst bringing about light production. As a consequence of ATP reaction with uciferin and Luciferase, a complex of adenine luciferin and inorganic pyrophosphate is produced through which the complex in the presence of oxygen leads to the production of light and the release of carbon dioxide [ 26 ]. In the next step, 100 µl of MgSO4 (100 mmol), 100 µl of ATP (40 mmol) and 400 µl of luciferin (5 mmol) were weighed and then dissolved in Tris buffer (50 mmol) with PH 7/8, and finally, it was brought to a volume of one ml with a standard buffer [ 26 ]. After preparing the essential material, all steps were performed in the presence of ice due to the sensitivity of ATP to temperature. Therefore, five microliters of cellular lysis solution entered the luminometer tube and then five microliters of a complex containing tris buffer, luciferin and MgSO4 were added. Finally, five microliters of luciferase enzyme entered the system. After several pipetting, it was inserted into the luminometer device, and the numbers were recorded at intervals of zero and 30 seconds [ 29 ]. It is worth pointing out that a standard curve would be essential for ATP measurement in the samples. Thus, ATP was first weighed, and its stock was completely dissolved in 50 mM Tris buffer with a PH of 8.7. The concentration of this stock was precisely calculated. It was used as the main stock to prepare a series of different concentrations of 1/2 for bioluminescence readings. In the following, the concentration of ATP was acquired [ 26 – 30 ]. Chemiluminescence Chemiluminescence is another method of luminescence that shows the chemical reaction by the device and is usually in the form of blue light. The basis of this method is typically the oxidation of luminol (C8H7N3O2) and its reaction with the sampl which was used in the present study. In the presence of an aprotic bipolar solution such as oxygen-containing dimethyl sulfoxide along with relatively strong oxidant (in most cases H2O2) and a suitable catalyst such as a metal ion or a type of 3-amino-phthalate oxidoreductase is excited. It would be returned to the steady-state by emitting light [ 31 ]. For this purpose, 60 µl of luminol (final length 0.03 mM) to 100 with 20 µl of H2O2 (0.001mM) were injected into sperm supernatant in the system wells (96 well anti-light micro plated). Then the amount of ROS was measured using Luminometer. Data was measured and recorded in RLU / s in 60 seconds [ 31 ]. Experimental groups Treatments At a concentration of 10 5 sperm cells/ml, male gametes were exposed to different dosages (high, medium, and low) of CP and NCP BVDV in an incubator at 38.5°C for 2 hours. CP and NCP BVDV with 3 different doses of 10 5 (high dose), 10 4 (medium dose), and 10 3 (low dose) tissue culture infectious dose (TCID) 50/mL were challenged to sperm cells. Viability, plasma membrane integrity, motility, oxidative stress, and apoptosis were all measured in the samples. Control The viability, membrane integrity, motility, oxidative stress, and apoptosis of a sperm sample with a concentration of 10 5 sperm per milliliter without BVDV biotypes were assessed. All of the above treatments and control group tests were carried out three times at various times. Statistical analysis Data were analyzed using GLM procedure. All analyses were conducted in SAS version 9.4 (SAS Institute Inc., Carry, NC, USA). Differences at P ≤ 0.05 were considered significant. Results Viability, membrane integrity and motility The results of the Eosin Nigrosine test (to investigate the survival rate of sperm after exposure to different virus biotypes), HOST (to evaluate the integrity of the sperm membrane after exposure to different virus biotypes) and CASA (to assess the motility parameters) revealed a significant difference (P ≤ 0.05) between both virus biotypes in comparison with the control group [ 24 ]. It was shown that the number of live sperm in the control group had a range of 72 ± 3.60 percent. At the same time, in both treatment groups, this value drops significantly when virus concentration rises (P ≤ 0.05). Also, the effect of virus biotypes on plasma membrane integrity has led to a significant drop in both high (10 5 ) and low (10 3 ) CP and NCP BVDV concentrations (P ≤ 0.05) [ 24 ]. Oxidative Stress and Apoptosis ROS The results of measurement of ROS released after exposure of BVD virus biotypes to sperm samples by chemiluminescence method showed a significant effect (P ≤ 0.05) of CP biotype on induction of oxidative stress in bovine spermatozoa. It should be highlighted that the impact of the high dose of CP biotype compared to the average and low dose of the same biotype was significant (P ≤ 0.05), but no significant difference was observed between the effect of medium and low dose. Also, none of the NCP biotype doses had a significant impact on increasing ROS production in spermatozoa (Fig. 1 ). Caspase 3/7 The results of measuring caspase 3/7 produced in interaction of BVD virus biotypes with sperm cells by Bioluminescence method showed a significant effect (P ≤ 0.05) of CP and NCP biotypes on the induction of planned cell death in bovine spermatozoa. It should be mentioned that the high dose impact of CP biotype compared to the average dose and the average dose to the low was significant (P ≤ 0.05). However, there was no significant difference between the effects of NCP biotype high, medium and low dosages (Fig. 2 ). Caspase 9 The results of measuring caspase 9 produced after exposure of BVD virus biotypes to sperm samples by the Bioluminescence method showed a significant effect (P ≤ 0.05) of the three dosages of CP in addition to the high dose of NCP upon the induction of planned cell death in bovine spermatozoa. Furthermore, the effect of the high dose of CP biotype compared with the average dose and the average dose with the low dose were significant. (P ≤ 0.05) (Fig. 3 ). ATP The results of measuring ATP produced after exposure of BVD virus biotypes to sperm samples by Bioluminescence method showed a significant effect (P ≤ 0.05) of the three dosages of CP and NCP on the induction of planned cell death in bovine spermatozoa. It is noteworthy that the effect of the high dose of CP compared to the medium dose and the medium dose to the low dose were significant (P ≤ 0.05). Nevertheless, no significant differences were indicated between the medium and low doses of NCP (Fig. 4 ). Discussion The effects of CP and NCP BVDV biotypes on bovine sperm cell viability, plasma membrane integrity, motility, and the production of oxidative stress and programmed cell death (apoptosis) in vitro were examined in previous [ 24 ] and the present study. The molecular mechanism of virus entrance into sperm cells is poorly understood. However, some articles have mentioned the importance of glycoproteins 48 and 53 (gp 48 and gp 53 ) on the virus membrane for attaching to host cells [ 32 ]. CD 46 molecules have been identified as BVDV receptors [ 33 ]. According to the findings, both virus biotypes substantially affected the survival, membrane integrity, and motility parameters of bovine sperm cells in vitro . One of the reasons for the decline in sperm survival after exposure to the CP and NCP BVDV biotypes could be the occurrence of programmed cell death (apoptosis) [ 34 ]. The intracellular ROS can be generated from damaged or weak sperm. More precisely, sperm derived from abnormal spermatogenesis can produce too much ROS. Although ROS is made in natural enzymatic reactions, enzymatic and non-enzymatic antioxidants would prevent cell damage [ 21 ]. The oxidant-antioxidant balance would simultaneously control the beneficial oxidants for regular cell function, in addition to, preventing cells from excessive oxidative stress damage. Semen consists of a high concentration of antioxidants as well as scavengers [ 36 ]. These antioxidants are sperm protectors from the harmful effects of ROS. Enzymatic antioxidants include superoxide dismutase, catalyze, glutathione peroxidase and serum semolina. On the other hand, Non-enzymatic antioxidants comprise albumin, barbotine, L-carnitine, glutathione, pyruvate, taurine, hypotaurine, vitamin E, C and zinc. The most striking intracellular protector against ROS would be glutathione [ 35 ]. This antioxidant contains a sulfhydryl group that collects free radicals directly. Laboratory studies reported that a decent balance between ROS and antioxidants would be required for fertilization [ 21 ]. H 2 O 2 can increase the potency and phosphorylation of tyrosine in vitro , whereas the catalyze enzyme would prevent that. Moreover, physiological concentrations of NO causes hyperactivity, capacitance, and zona pellucida attachment, while its excessive concentrations would have inhibitory effects [ 21 ], the balance between antioxidants and ROS would be a prerequisite for chromatin aggregation in maturing sperm during epididymis transmission. Improper chromatin aggregation while sperm reaches the epididymis tail would contribute to abnormal morphology, sperm dysfunction, and infertility [ 23 ]. One of the first functions affected by oxidative stress and lipid peroxidation is sperm motility. The correlation between peroxide lipid formation and sperm motility in the ROS production system has been observed many times. The sensitivity of sperm motility to oxidative attack has been indicated [ 36 ]. Even though the effective mechanisms in reducing sperm motility when exposed to oxidative stress are still unknown, it appears that both oxidative damages to the axoneme and reduction of intracellular ATP are involved in this process [ 23 ]. Given the dramatic effects of high levels of ROS on sperm motility, it is evident that oxidative stress can also reduce the fertility of normal sperm. In this case, the sperm's capacity to attach to the oocyte's membrane is impaired. On the other hand, in the lower levels of oxidative stress, increased sperm and oocyte attachment is occurred that is possibly due to the 1) positive role of ROS in tyrosine phosphorylation associated with sperms capacity and 2) The importance of sterol oxidation in facilitating the removal of cholesterol from sperm membrane (Dutta et al., 2019). Nevertheless, in the higher levels of oxidative stress, the induction of lipid peroxidation in the plasma membrane is associated with reduced sperm and oocyte attachment, perhaps owing to the direct induction of oxidative damage to the proteins involved in the process of binding sperm and oocyte. The impaired function of sperm that is often induced by oxidative stress can affect the motility of these cells, DNA integration, and the binding of sperm to the oocyte [ 22 ]. It seems that suppressed sperm motility by oxidative stress is directly related to the induction of lipid peroxidation. When ROS attacks unsaturated fatty acids that are more abundantly found in the sperm membrane than other cells, various fatty metabolites, including proxyl lipids, alkyl radicals, and various aldehydes, will be produced [ 36 ](Aitken 2020). The addition of peroxide lipids and lipid aldehydes to the human sperm population leads to a rapid decrease in the motility of these cells through distinct mechanisms. The decline in motility may be related to the steps that ultimately lead to a reduction in axoneme protein phosphorylation and sperm immobilization, both of which are associated with decreased membrane fluidity. It is noteworthy that membrane fluidity is essential for the attachment of sperm to the oocyte membrane [ 36 ]. Therefore, on top of the effect of peroxidation on the damage of lipid membrane structure, it also affects the ability of sperm to participate in processes associated with the oocyte membrane. Sperm parameters such as concentration, motility, and morphology are commonly used to determine sperm fertilization potential. DNA strands can be a source of reproductive potential between fertile and infertile animals [ 22 ]. It has also been reported that chromatin in the sperm nucleus is vulnerable to oxidative stress and leads to DNA changes and fragmentation [ 22 ]. Oxidative stress is known as a major cause of DNA damage in sperm. In numerous studies, decreased antioxidant capacity in addition to the high concentration of ROS in the semen of DNA-damaged animals has been identified. DNA repair is limited in sperm and occurs only at certain stages of spermatogenesis. Although the repair mechanisms in nuclear density progress are not active in the epididymis, spermatozoa in the epididymis and during transmission in semen are in exposure to oxidative damage. The oocyte is the next opportunity for DNA repair, which is an important step in fetal development. Nevertheless, oxidative stress-induced DNA compounds may be recoverable by oocytes through the breakdown in one or both strands of DNA may not be regenerated and may ultimately hurt fertility and pregnancy outcomes [ 22 ]. Another theory about DNA damage of sperm besides impaired fertility is programmed cell death or apoptosis [ 22 ]. Apoptosis is a physiological phenomenon through which cellular morphological and biochemical changes occur, resulting in programmed cell death. In somatic cells, apoptosis is associated with extensive nuclear fragmentation due to mitochondrial-released nuclease (including endonuclease G) or activated in the cystocele (e.g., caspase-activating DNAase) [ 35 ]. However, the sperm's mitochondria and the major part of its cytoplasm are located in the middle part of the sperm. They are totally separated from the nucleus, making it special from any other cell type in biology. Thus when apoptosis is activated by PI 3 kinase inhibitor, the nuclease remains in the middle part and cannot penetrate the nucleus. If apoptosis is induced in sperm cells, the DNA cannot be cleaved by nuclease. The only apoptotic product that can harm the DNA of sperm is ROS produced by mitochondria. Mitochondria are powerful generators of ROS in sperm, and this activity would increase the apoptosis induction. This is why the most damage seen in sperm DNA is naturally due to the oxidative process [ 22 ]. Conclusion It is concluded that CP and NCP biotypes of BVDV can affect the vital aspects, dynamic features and also induction of oxidative stress and apoptosis in interaction with male bovine gamete. Declarations Acknowledgments We would like to thank the vice-chancellor for Research and Technology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper. Funding information This work was supported by the Research Council of Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran. Data availability The data used in this study can be available from the corresponding authors upon reasonable request. Ethics approval and consent to participate Protocol of this study was confirmed by animal welfare committee of University of Tehran, Faculty of Veterinary Medicine, Veterinary Ethical Review Committee (https://ethics.research.ac.ir/IR.UT.VETMED.REC.1404.015) in accordance with institutional and national or international guidelines. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical trial number Not applicable References Childs T. X. Disease of Cattle—Saskatchewan. Can. J. Comp. Med. Vet. Sci. 1946. 10(11):316. Olafson P. 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Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation. Vet. Microbil. 2009. 134(1-2):106-12. Mehrzad J, Malvandi AM, Alipour M, Hosseinkhani S. Environmentally relevant level of aflatoxin B1 elicits toxic pro-inflammatory response in murine CNS-derived cells. Toxicol. Lett. 2017. 279:96-106. Mehrzad J, Fazel F, Pouyamehr N, Hosseinkhani S, Dehghani H. Naturally occurring level of aflatoxin B1 injures human, canine and bovine leukocytes through ATP depletion and caspase activation. IJT. 2020. 39(1):30-8. Mehrzad J, Hosseinkhani S, Malvandi AM. Human microglial cells undergo proapoptotic induction and inflammatory activation upon in vitro exposure to a naturally occurring level of aflatoxin B1. Neuroimmunomodulation. 2018. 5(3):176-83. Barni F, Lewis SW, Berti A, Miskelly GM, Lago G. Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta. 2007.72(3):896-913. Garoussi MT, Mehrzad J. Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle. Theriogenology. 2011. 75(6):1067-75. Maurer K, Krey T, Moennig V, Thiel H-Jr, Rümenapf T. CD46 is a cellular receptor for bovine viral diarrhea virus. J. Virol. 2004. 78(4):1792-9. Givens MD, Marley MS. Immunology of chronic BVDV infections. Biologicals. 2013.41(1):26-30. Drevet JR, Aitken R. Oxidative damage to sperm DNA: attack and defense. Genetic Damage in Human Spermatozoa: Springer. p. 2019. 107-17. Aitken RJ. Impact of oxidative stress on male and female germ cells: implications for fertility. Reprod. 2020. 159(4): R189-R201. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 Mar, 2026 Reviews received at journal 15 Feb, 2026 Reviewers agreed at journal 06 Feb, 2026 Editor invited by journal 11 Jan, 2026 Reviews received at journal 30 Nov, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers agreed at journal 19 Sep, 2025 Reviewers invited by journal 17 Sep, 2025 Editor assigned by journal 14 Aug, 2025 Submission checks completed at journal 13 Aug, 2025 First submitted to journal 13 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7160788","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":517808643,"identity":"1e37235f-e6bf-480d-8c89-eca24c4295b1","order_by":0,"name":"Mehran Dabiri","email":"","orcid":"","institution":"University of Technology Sydney. Sydney","correspondingAuthor":false,"prefix":"","firstName":"Mehran","middleName":"","lastName":"Dabiri","suffix":""},{"id":517808644,"identity":"4c4ae220-c6f5-427b-97cf-23c90e19a907","order_by":1,"name":"Massoud Talebkhan Garoussi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBADHn4Yg3gtkg3MJGphMDjATKRKefezDx/z/KmTMb6Rf4DhRw2DjHkDAS2GZ9KNjXnbDvOY3UhmYOw5xsAjc4CQloY0NmnehgNgLQy8DQw8EoQcZtj/jE0a6DAe4xlAW/4So0VeAmgLDxszj4FEMgMzUbYYSDxjNpwL9IvEmccGh2WOSRBhS38a44M3f+rs+dsTHz58U2NjT9iWA0gcIJugBqAtDYTVjIJRMApGwUgHAAj9MbzZQExHAAAAAElFTkSuQmCC","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Massoud","middleName":"Talebkhan","lastName":"Garoussi","suffix":""},{"id":517808645,"identity":"028124a2-d9f7-4321-b5d9-2a9bc04431d5","order_by":2,"name":"Jalil Mehrzad","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Jalil","middleName":"","lastName":"Mehrzad","suffix":""},{"id":517808646,"identity":"599d0775-7aff-4c08-aaa6-b1e7968c987c","order_by":3,"name":"Parviz Tajik","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Parviz","middleName":"","lastName":"Tajik","suffix":""},{"id":517808647,"identity":"a37cee9f-47ed-4bd4-97ce-0b0cb2591975","order_by":4,"name":"Sara Hosseini","email":"","orcid":"","institution":"Bu-Ali Research Institute, Mashhad University of Medical","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Hosseini","suffix":""}],"badges":[],"createdAt":"2025-07-18 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17:30:51","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100709,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7160788/v1/2e7e07c1f38a33fe66acc028.html"},{"id":92202338,"identity":"8a2d3bf3-0d7e-49c9-9da4-3d2f3a0e3c84","added_by":"auto","created_at":"2025-09-25 17:30:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64529,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of ROS in interaction of CP and NCP BVDV biotypes with bovine sperm cells using Chemiluminescence. CP BVDV biotype impact significantly (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7160788/v1/f355fac53429773a6252b6df.png"},{"id":92202337,"identity":"ebb4c0d2-f894-4c96-9d5a-e57a8f36a89a","added_by":"auto","created_at":"2025-09-25 17:30:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65261,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase 3/7 produced in interaction of Bovine sperm cells with BVDV biotypes in apoptosis of male gamete.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7160788/v1/2bf5499784c97ce9ef5f7bd7.png"},{"id":92202340,"identity":"1b995aa4-ddd1-4dc2-ab4f-73f93e29d15a","added_by":"auto","created_at":"2025-09-25 17:30:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66645,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase 9 produced in interaction of bovine sperm cells with BVDV biotypes in apoptosis of male gamete.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7160788/v1/4becdc65805c6ce67592ede4.png"},{"id":92203122,"identity":"6cbf9426-8a4f-400d-b4fa-afc9b7b8fe9b","added_by":"auto","created_at":"2025-09-25 17:38:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":66914,"visible":true,"origin":"","legend":"\u003cp\u003eATP produced in interaction of BVD virus CP and NCP biotypes with Holstein sperm cells. the impact of the high dose of CP compared to the medium dose and the medium dose to the low dose were significant (P £ 0.05).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7160788/v1/7e2c172f16cdb3bbed5656bb.png"},{"id":92204333,"identity":"1b820115-0e8e-4772-a93a-dd8d593fca36","added_by":"auto","created_at":"2025-09-25 18:02:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":935406,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7160788/v1/3d31db96-3dbc-476b-b527-8b0662b969ff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interaction of CP and NCP BVDV with bovine sperm cells on apoptosis and oxidative stress in vitro","fulltext":[{"header":"Introduction","content":"\u003cp\u003eContrary to its name, which refers to diarrhea, the bovine viral diarrhea virus (BVDV) is among the most dangerous viral illnesses in cattle and other ruminants, exhibiting extensive detrimental consequences on the reproductive tract and fertility. This pathogen was first discovered in 1946 and is one of the germs which can cause substantial economic losses to the dairy industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In immune hosts, the illness is subclinical, also 70 to 90% of animals with acute form do not show clinical manifestations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. BVDV is the first member of the \u003cem\u003eFlaviviridae\u003c/em\u003e family and is a single-stranded, enveloped RNA virus. The virus is currently divided into four strains: BVDV genotypes 1 and 2, borderline disease virus (BDV), and classical swine fever virus (CSFV). BVDV isolates can be classified into Cytopathic (CP) and Non-Cytopathic (NCP) biotypes based on their disastrous impacts on the cells. CP isolates, unlike NCP, can trigger vacuole formation in the cytoplasm along with cell death in the culture medium [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. NCP biotypes are more abundant in nature and comprise 60 to 90% of isolates. Only NCP biotype strains can cause chronic infection. These strains are assumed to be a common source of CP strains because of a certain nucleotide combination in the NS2-3 genomic region [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe BVDV can grow in both male and female gonads, along with all sections of the female reproductive tract. BVDV infection in cattle can be fatal during pregnancy, especially in the pre-trimester stage, which is riskier. Both biotypes of the virus can infect the fetus and easily penetrate the placental barrier [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. BVDV's natural host is cattle and the illness is endemic in cattle herds around the world [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to serological surveys conducted in various geographic regions, the prevalence of BVDV infection cattle ranges from 40 to 90 percent in individual cattle and 28 to 66 percent in cattle herds, with 0.5 to 2.5 percent of cattle persistently infected (PI) with the virus [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. BVDV is found in more than 50 to 100 percent of dairy herds in Iran, according to epidemiological studies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDirect contact between animals is the most typical way for BVDV to spread [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. BVDV is shed in various bodily fluids and excretions by infected cattle, including nasal discharge, saliva, sperm, urine, feces, tears, milk, and uterine flushing [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. BVDV can also be spread during rectal examinations and also during natural breeding or artificial insemination (AI) of cattle with infected bulls' sperm [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBVDV can infect male reproductive organs, and researchers are looking into the effects of virus infection on testicular function and bull fertility in many studies. Infection of sperm with this agent might come from four different places: 1) Permanent infection of male fetus throughout gestation (vertical transmission), 2) Acute infection of the bull following development of immune system, 3) Chronic infection of the testicle after protracted acute infection, and 4) Persistent testicular infection out of an unidentified source [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition to, infection of \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e embryos can be accomplished in three ways: 1) donor cows, 2) recipient animals, and 3) animal-based laboratory products such as bovine fetal serum (BFS). Each of these factors has the potential to influence agent transmission. Following both acute and persistent infection, seminal vesicles and the prostate gland can be an ideal refuge for BVDV multiplication and subsequent transfer to the semen of bulls [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA few studies found that neither acutely infected nor PI bulls had any evident changes in sperm quality or semen. Several investigations, on the other hand, found irregularities such as decreased sperm volume, disrupted sperm concentration and motility, and increased sperm malformations [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Cows bred with PI bull's sperm had a 38 percent conception rate compared to 66 percent for those bred with uninfected bull's sperm [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As a result, BVDV infection can affect testicular activity, resulting in spermatozoa anomalies. Semen from these sick bulls could be a cause of infection for sensitive cows, lowering conception rates and fertility in cows after natural breeding or artificial insemination.\u003c/p\u003e\u003cp\u003eBecause there isn't enough intracytoplasmic space for defense enzymes and antioxidants to concentrate, spermatozoa are extremely sensitive to oxidative damage. Furthermore, the plasma membrane's high amount of unsaturated fatty acids renders it more vulnerable to oxidative stress [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Meanwhile, sperm capacitation generates a lot of reactive oxygen species (ROS), which could also lead to oxidative stress and subsequent functional failure. The cell can be driven to programmed cell death (apoptosis) under these conditions, which is followed by mitochondrial ROS production, depletion of mitochondrial membrane potential, caspase activity, phosphatidylserine overexpression, and DNA oxidative injury [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs a result, any condition that causes an increase in the production of these chemicals in sperm can have an impact on its performance. Disrupting critical elements of the sperm, such as the plasma membrane, mitochondria, or acrosomes, might induce this unfavorable effect. As a result, measuring ROS and apoptotic indicators can reveal how the BVDV mechanism harms bovine spermatozoa [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe goal of this study was to see how CP and NCP BVDV affected the performance of Holstein bull spermatozoa \u003cem\u003ein vitro\u003c/em\u003e. Viability, membrane integrity, motility, viral-induced oxidative stress, and apoptosis were all measured in the spermatozoa.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBVDV free sperm samples\u003c/h2\u003e\u003cp\u003eAll utilized sperm samples were checked by PCR before any tests to rule out the existence of the BVD virus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The BVD virus was not found in any of the sperm sample.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSperm samples\u003c/h3\u003e\n\u003cp\u003eBVDV-free bull's frozen sperm which were routinely used in farms for Artificial Insemination (AI) of dairy cows was thawed in a water bath at 37\u0026deg;C and added to the head of a Percoll gradient (45 and 90 percent; Pharmacia, Uppsala, Sweden). The following method was used to distinguish live and dead spermatozoa: the sperm sample was centrifuged for 30 minutes at 2000 g. After centrifugation, the supernatant was removed, and the sperm pellet was re-suspended in TALP\u0026thinsp;+\u0026thinsp;BSA (bovine serum albumin; Sigma-Aldrich, Bornem, Belgium). The sperm pellet was then re-suspended to the desired concentration of 10\u003csup\u003e5\u003c/sup\u003e sperm/ml. frozen sperm samples were taken from a serial number of the national breeding facility for animal production enhancement (Karaj, Iran), which were BVDV-free.\u003c/p\u003e\n\u003ch3\u003eVirus\u003c/h3\u003e\n\u003cp\u003eHigh dose (10\u003csup\u003e5\u003c/sup\u003e), medium dose (10\u003csup\u003e4\u003c/sup\u003e), and low dose (10\u003csup\u003e3\u003c/sup\u003e) (TCID50 / ml) CP and NCP biotypes were utilized. The virus was cultivated in a 5 percent fetal calf serum Minimum Essential Medium (MEM) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eViability, membrane integrity and motility evaluation\u003c/h3\u003e\n\u003cp\u003eViability, membrane integrity and motility were assessed according to the prior experiment performed by the current team [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBioluminescence Experiment on Sperms\u003c/h3\u003e\n\u003cp\u003eThe generation and emission of light by a living creature is known as bioluminescence. Luciferase is a frequent enzyme involved in this process. This approach was used to assess the activity of caspase 3 and 7, caspase 9, and ATP in this investigation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis test was carried out with the aid of an illuminometer. Before beginning the test, the pipes would have been thoroughly cleaned and dried with distilled water and alcohol, resulting in a relative light unit (RLU) of less than 300 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Luciferase\u003c/h2\u003e\u003cp\u003eThe Luciferase enzyme, synthesized in Tarbiat Modares University- Iran, was employed in this study according to the following protocol: The bacteria BL21, which contains this enzyme was used. To be more precise, Firstly, Lactose or IPTG was added to a plasmid expressing Luciferase to expand the expression. Secondly, it was purified on the Ni-NTA nickel sepharose chromatography column. Lastly, one milliliter of elution (protein collected from the column) along with 120 microliters of 60% glycerol was stored in the freezer to be used in later molecular experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell Lysis\u003c/h3\u003e\n\u003cp\u003eIn terms of evaluating the activity of Caspase and ATP, the sperm pellet must primarily be lysed. For this purpose, cell culture lysis buffer (CCLR) was applied. The instruction of preparing this buffer is that 10ml glycerol, 231mg potassium dihydrogen phosphate salt (KH2PO4), 37 mg EDTA, 3.18 gr dipotassium mono-hydrogen phosphate, Triton 1%, and 48 \u0026micro;l double Mercaptoethanol was dissolved in deionized distilled water, and the magnet was placed in a tube and stirred and at last made its volume to 100 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the presence of ice, 150 \u0026micro;l of CCLR buffer was added to help the sperm pellet lyse. After 50 times of gentle pipetting, they were placed in the shaker section of the refrigerator for 30 minutes to complete dissolution. In the next step, it was centrifuged at 4\u0026deg;C for 10 minutes at 3000 rpm. The obtained solution was evenly distributed in three microtubes for bioluminescence experiments [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eThe Measurement of Caspase 3 and 7 in Sperms\u003c/h3\u003e\n\u003cp\u003eIn this experiment, sperm Caspases 3 and 7 against Luciferase enzyme were assessed. These components of the Caspase family play a key role in cellular apoptosis. This method is based on luminogenic Caspase 3 and 7 substrates (containing DEVD tetrapeptide sequence). It implies that following the breakdown of caspases 3 and 7, the luciferase substrate (amino-luciferin) is released, which results in reaction with Luciferase and consequently production/emission of light.\u003c/p\u003e\u003cp\u003eIn this section, three microliters of sperm lysis were predominantly added to the bioluminescence tubes. Two microliters of luciferase enzyme were added, and at times of zero, five, 10, 15, 20, 25 minutes, the producing bioluminescence was read and recorded in terms of RLU/s by the Illuminator device [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eThe Measurement of Caspase 9 in Sperms\u003c/h2\u003e\u003cp\u003eIn this section, the activity of Caspase 9 was evaluated. This enzyme plays a crucial role in the innate pathway of apoptosis. This method, which is a homogeneous luminescent test, is based on the luminogenic substrate of caspase 9 (with the LEHD tetrapeptide sequence), which is released as a consequence of the breakdown of caspase 9 by the reaction of luciferase substrate (amino-luciferin) with Luciferase, resulting in light production [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this part, at first, five microliters of cell lysis were added to the bioluminescence tubes. Two microliters of luciferase enzyme were added to it. Every 30 seconds in 0\u0026ndash;10 minutes, the numbers are shown as RLU / s were recorded by the device and the maximum absorbance was reported during this period [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eThe Measurement of ATP\u003c/h2\u003e\u003cp\u003eATP measurement would be another factor to determine sperm cell damage. This method was used for very low concentrations of ATP, which is based on the reaction of ATP with Luciferase and the oxidation of the luciferin catalyst bringing about light production. As a consequence of ATP reaction with uciferin and Luciferase, a complex of adenine luciferin and inorganic pyrophosphate is produced through which the complex in the presence of oxygen leads to the production of light and the release of carbon dioxide [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the next step, 100 \u0026micro;l of MgSO4 (100 mmol), 100 \u0026micro;l of ATP (40 mmol) and 400 \u0026micro;l of luciferin (5 mmol) were weighed and then dissolved in Tris buffer (50 mmol) with PH 7/8, and finally, it was brought to a volume of one ml with a standard buffer [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAfter preparing the essential material, all steps were performed in the presence of ice due to the sensitivity of ATP to temperature. Therefore, five microliters of cellular lysis solution entered the luminometer tube and then five microliters of a complex containing tris buffer, luciferin and MgSO4 were added. Finally, five microliters of luciferase enzyme entered the system. After several pipetting, it was inserted into the luminometer device, and the numbers were recorded at intervals of zero and 30 seconds [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt is worth pointing out that a standard curve would be essential for ATP measurement in the samples. Thus, ATP was first weighed, and its stock was completely dissolved in 50 mM Tris buffer with a PH of 8.7. The concentration of this stock was precisely calculated. It was used as the main stock to prepare a series of different concentrations of 1/2 for bioluminescence readings. In the following, the concentration of ATP was acquired [\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eChemiluminescence\u003c/h2\u003e\u003cp\u003eChemiluminescence is another method of luminescence that shows the chemical reaction by the device and is usually in the form of blue light. The basis of this method is typically the oxidation of luminol (C8H7N3O2) and its reaction with the sampl which was used in the present study. In the presence of an aprotic bipolar solution such as oxygen-containing dimethyl sulfoxide along with relatively strong oxidant (in most cases H2O2) and a suitable catalyst such as a metal ion or a type of 3-amino-phthalate oxidoreductase is excited. It would be returned to the steady-state by emitting light [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor this purpose, 60 \u0026micro;l of luminol (final length 0.03 mM) to 100 with 20 \u0026micro;l of H2O2 (0.001mM) were injected into sperm supernatant in the system wells (96 well anti-light micro plated). Then the amount of ROS was measured using Luminometer. Data was measured and recorded in RLU / s in 60 seconds [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eExperimental groups\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003eTreatments\u003c/h2\u003e\u003cp\u003eAt a concentration of 10\u003csup\u003e5\u003c/sup\u003e sperm cells/ml, male gametes were exposed to different dosages (high, medium, and low) of CP and NCP BVDV in an incubator at 38.5\u0026deg;C for 2 hours. CP and NCP BVDV with 3 different doses of 10\u003csup\u003e5\u003c/sup\u003e (high dose), 10\u003csup\u003e4\u003c/sup\u003e (medium dose), and 10\u003csup\u003e3\u003c/sup\u003e (low dose) tissue culture infectious dose (TCID) 50/mL were challenged to sperm cells. Viability, plasma membrane integrity, motility, oxidative stress, and apoptosis were all measured in the samples.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eControl\u003c/h2\u003e\u003cp\u003eThe viability, membrane integrity, motility, oxidative stress, and apoptosis of a sperm sample with a concentration of 10\u003csup\u003e5\u003c/sup\u003e sperm per milliliter without BVDV biotypes were assessed.\u003c/p\u003e\u003cp\u003eAll of the above treatments and control group tests were carried out three times at various times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using GLM procedure. All analyses were conducted in SAS version 9.4 (SAS Institute Inc., Carry, NC, USA). Differences at P\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eViability, membrane integrity and motility\u003c/h2\u003e\u003cp\u003eThe results of the Eosin Nigrosine test (to investigate the survival rate of sperm after exposure to different virus biotypes), HOST (to evaluate the integrity of the sperm membrane after exposure to different virus biotypes) and CASA (to assess the motility parameters) revealed a significant difference (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) between both virus biotypes in comparison with the control group [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt was shown that the number of live sperm in the control group had a range of 72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60 percent. At the same time, in both treatment groups, this value drops significantly when virus concentration rises (P\u0026thinsp;\u0026le;\u0026thinsp;0.05). Also, the effect of virus biotypes on plasma membrane integrity has led to a significant drop in both high (10\u003csup\u003e5\u003c/sup\u003e) and low (10\u003csup\u003e3\u003c/sup\u003e) CP and NCP BVDV concentrations (P\u0026thinsp;\u0026le;\u0026thinsp;0.05) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eOxidative Stress and Apoptosis\u003c/h2\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003eROS\u003c/h2\u003e\u003cp\u003eThe results of measurement of ROS released after exposure of BVD virus biotypes to sperm samples by chemiluminescence method showed a significant effect (P \u0026le; 0.05) of CP biotype on induction of oxidative stress in bovine spermatozoa. It should be highlighted that the impact of the high dose of CP biotype compared to the average and low dose of the same biotype was significant (P \u0026le; 0.05), but no significant difference was observed between the effect of medium and low dose. Also, none of the NCP biotype doses had a significant impact on increasing ROS production in spermatozoa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eCaspase 3/7\u003c/h2\u003e\u003cp\u003eThe results of measuring caspase 3/7 produced in interaction of BVD virus biotypes with sperm cells by Bioluminescence method showed a significant effect (P \u0026le; 0.05) of CP and NCP biotypes on the induction of planned cell death in bovine spermatozoa. It should be mentioned that the high dose impact of CP biotype compared to the average dose and the average dose to the low was significant (P \u0026le; 0.05). However, there was no significant difference between the effects of NCP biotype high, medium and low dosages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eCaspase 9\u003c/h2\u003e\u003cp\u003eThe results of measuring caspase 9 produced after exposure of BVD virus biotypes to sperm samples by the Bioluminescence method showed a significant effect (P \u0026le; 0.05) of the three dosages of CP in addition to the high dose of NCP upon the induction of planned cell death in bovine spermatozoa. Furthermore, the effect of the high dose of CP biotype compared with the average dose and the average dose with the low dose were significant. (P \u0026le; 0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eATP\u003c/h2\u003e\u003cp\u003eThe results of measuring ATP produced after exposure of BVD virus biotypes to sperm samples by Bioluminescence method showed a significant effect (P \u0026le; 0.05) of the three dosages of CP and NCP on the induction of planned cell death in bovine spermatozoa. It is noteworthy that the effect of the high dose of CP compared to the medium dose and the medium dose to the low dose were significant (P \u0026le; 0.05). Nevertheless, no significant differences were indicated between the medium and low doses of NCP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe effects of CP and NCP BVDV biotypes on bovine sperm cell viability, plasma membrane integrity, motility, and the production of oxidative stress and programmed cell death (apoptosis) \u003cem\u003ein vitro\u003c/em\u003e were examined in previous [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and the present study.\u003c/p\u003e\u003cp\u003eThe molecular mechanism of virus entrance into sperm cells is poorly understood. However, some articles have mentioned the importance of glycoproteins 48 and 53 (gp\u003csup\u003e48\u003c/sup\u003e and gp\u003csup\u003e53\u003c/sup\u003e) on the virus membrane for attaching to host cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. CD\u003csup\u003e46\u003c/sup\u003e molecules have been identified as BVDV receptors [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. According to the findings, both virus biotypes substantially affected the survival, membrane integrity, and motility parameters of bovine sperm cells \u003cem\u003ein vitro\u003c/em\u003e. One of the reasons for the decline in sperm survival after exposure to the CP and NCP BVDV biotypes could be the occurrence of programmed cell death (apoptosis) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe intracellular ROS can be generated from damaged or weak sperm. More precisely, sperm derived from abnormal spermatogenesis can produce too much ROS. Although ROS is made in natural enzymatic reactions, enzymatic and non-enzymatic antioxidants would prevent cell damage [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The oxidant-antioxidant balance would simultaneously control the beneficial oxidants for regular cell function, in addition to, preventing cells from excessive oxidative stress damage. Semen consists of a high concentration of antioxidants as well as scavengers [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These antioxidants are sperm protectors from the harmful effects of ROS. Enzymatic antioxidants include superoxide dismutase, catalyze, glutathione peroxidase and serum semolina. On the other hand, Non-enzymatic antioxidants comprise albumin, barbotine, L-carnitine, glutathione, pyruvate, taurine, hypotaurine, vitamin E, C and zinc. The most striking intracellular protector against ROS would be glutathione [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This antioxidant contains a sulfhydryl group that collects free radicals directly. Laboratory studies reported that a decent balance between ROS and antioxidants would be required for fertilization [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e can increase the potency and phosphorylation of tyrosine \u003cem\u003ein vitro\u003c/em\u003e, whereas the catalyze enzyme would prevent that. Moreover, physiological concentrations of NO causes hyperactivity, capacitance, and zona pellucida attachment, while its excessive concentrations would have inhibitory effects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the balance between antioxidants and ROS would be a prerequisite for chromatin aggregation in maturing sperm during epididymis transmission. Improper chromatin aggregation while sperm reaches the epididymis tail would contribute to abnormal morphology, sperm dysfunction, and infertility [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. One of the first functions affected by oxidative stress and lipid peroxidation is sperm motility. The correlation between peroxide lipid formation and sperm motility in the ROS production system has been observed many times. The sensitivity of sperm motility to oxidative attack has been indicated [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Even though the effective mechanisms in reducing sperm motility when exposed to oxidative stress are still unknown, it appears that both oxidative damages to the axoneme and reduction of intracellular ATP are involved in this process [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Given the dramatic effects of high levels of ROS on sperm motility, it is evident that oxidative stress can also reduce the fertility of normal sperm. In this case, the sperm's capacity to attach to the oocyte's membrane is impaired. On the other hand, in the lower levels of oxidative stress, increased sperm and oocyte attachment is occurred that is possibly due to the 1) positive role of ROS in tyrosine phosphorylation associated with sperms capacity and 2) The importance of sterol oxidation in facilitating the removal of cholesterol from sperm membrane (Dutta et al., 2019). Nevertheless, in the higher levels of oxidative stress, the induction of lipid peroxidation in the plasma membrane is associated with reduced sperm and oocyte attachment, perhaps owing to the direct induction of oxidative damage to the proteins involved in the process of binding sperm and oocyte. The impaired function of sperm that is often induced by oxidative stress can affect the motility of these cells, DNA integration, and the binding of sperm to the oocyte [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It seems that suppressed sperm motility by oxidative stress is directly related to the induction of lipid peroxidation. When ROS attacks unsaturated fatty acids that are more abundantly found in the sperm membrane than other cells, various fatty metabolites, including proxyl lipids, alkyl radicals, and various aldehydes, will be produced [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e](Aitken 2020). The addition of peroxide lipids and lipid aldehydes to the human sperm population leads to a rapid decrease in the motility of these cells through distinct mechanisms. The decline in motility may be related to the steps that ultimately lead to a reduction in axoneme protein phosphorylation and sperm immobilization, both of which are associated with decreased membrane fluidity. It is noteworthy that membrane fluidity is essential for the attachment of sperm to the oocyte membrane [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, on top of the effect of peroxidation on the damage of lipid membrane structure, it also affects the ability of sperm to participate in processes associated with the oocyte membrane. Sperm parameters such as concentration, motility, and morphology are commonly used to determine sperm fertilization potential.\u003c/p\u003e\u003cp\u003eDNA strands can be a source of reproductive potential between fertile and infertile animals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It has also been reported that chromatin in the sperm nucleus is vulnerable to oxidative stress and leads to DNA changes and fragmentation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Oxidative stress is known as a major cause of DNA damage in sperm. In numerous studies, decreased antioxidant capacity in addition to the high concentration of ROS in the semen of DNA-damaged animals has been identified. DNA repair is limited in sperm and occurs only at certain stages of spermatogenesis. Although the repair mechanisms in nuclear density progress are not active in the epididymis, spermatozoa in the epididymis and during transmission in semen are in exposure to oxidative damage. The oocyte is the next opportunity for DNA repair, which is an important step in fetal development. Nevertheless, oxidative stress-induced DNA compounds may be recoverable by oocytes through the breakdown in one or both strands of DNA may not be regenerated and may ultimately hurt fertility and pregnancy outcomes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Another theory about DNA damage of sperm besides impaired fertility is programmed cell death or apoptosis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Apoptosis is a physiological phenomenon through which cellular morphological and biochemical changes occur, resulting in programmed cell death. In somatic cells, apoptosis is associated with extensive nuclear fragmentation due to mitochondrial-released nuclease (including endonuclease G) or activated in the cystocele (e.g., caspase-activating DNAase) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the sperm's mitochondria and the major part of its cytoplasm are located in the middle part of the sperm. They are totally separated from the nucleus, making it special from any other cell type in biology. Thus when apoptosis is activated by PI\u003csub\u003e3\u003c/sub\u003e kinase inhibitor, the nuclease remains in the middle part and cannot penetrate the nucleus. If apoptosis is induced in sperm cells, the DNA cannot be cleaved by nuclease. The only apoptotic product that can harm the DNA of sperm is ROS produced by mitochondria. Mitochondria are powerful generators of ROS in sperm, and this activity would increase the apoptosis induction. This is why the most damage seen in sperm DNA is naturally due to the oxidative process [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt is concluded that CP and NCP biotypes of BVDV can affect the vital aspects, dynamic features and also induction of oxidative stress and apoptosis in interaction with male bovine gamete.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the vice-chancellor for Research and Technology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interests regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Research Council of Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study can be available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtocol of this study was confirmed by animal welfare committee of University of Tehran, Faculty of Veterinary Medicine, Veterinary Ethical Review Committee (https://ethics.research.ac.ir/IR.UT.VETMED.REC.1404.015) in accordance with institutional and national or international guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChilds T. 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Rec. 1985.117(10):240-5.\u003c/li\u003e\n\u003cli\u003eWalz P, Grooms D, Passler T, Ridpath J, Tremblay R, Step D, Callan R.J., Givens M.D. Control of bovine viral diarrhea virus in ruminants. JVIM. 2010. 24(3):476-86.\u003c/li\u003e\n\u003cli\u003eAragaw K, Sibhat B, Ayelet G, Skjerve E, Gebremedhin EZ, Asmare K. (2018). Seroprevalence and factors associated with bovine viral diarrhea virus (BVDV) infection in dairy cattle in three milksheds in Ethiopia. Trop. Anim. Health Prod. 2018. 50(8):1821-7.\u003c/li\u003e\n\u003cli\u003eYeşilbağ K, Alpay G, Becher P. Variability and global distribution of subgenotypes of bovine viral diarrhea virus. Viruses. 2017. 9(6):128.\u003c/li\u003e\n\u003cli\u003eScharnb\u0026ouml;ck B, Roch F-F, Richter V, Funke C, Firth CL, Obritzhauser W, Baumgartner W, K\u0026auml;sbohrer A, Pinior B. A meta-analysis of bovine viral diarrhoea virus (BVDV) prevalences in the global cattle population. Sci. Rep. 2018. 8(1):1-15.\u003c/li\u003e\n\u003cli\u003eVelasova M, Damaso A, Prakashbabu BC, Gibbons J, Wheelhouse N, Longbottom D, Winden S, Green M, Guitian J. Herd-level prevalence of selected endemic infectious diseases of dairy cows in Great Britain. J. Dairy Sci. 2017. 100:9215\u0026ndash;9233\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eGaroussi M, Mehrzad J, Nejati A. Investigation of persistent infection of bovine viral diarrhea virus (BVDV) in Holstein dairy cows. Trop. Anim. Health Prod. 2019. 51(4):853-8.\u003c/li\u003e\n\u003cli\u003eThurmond, M. C. Virus transmission. Bovine viral diarrhea virus: Diagnosis, management and control Ames, IA: Blackwell Publishing. 2005.1: 91\u0026ndash;104.\u003c/li\u003e\n\u003cli\u003eLanyon SR, Hill FI, Reichel MP, Brownlie J. Bovine viral diarrhoea: pathogenesis and diagnosis. Vet. J. 2014. 199(2):201-9.\u003c/li\u003e\n\u003cli\u003eLang-Ree J, Vatn T, Kommisrud E, L\u0026oslash;ken T. Transmission of bovine viral diarrhoea virus by rectal examination. Vet. Rec. 1994;135(17):412-3.\u003c/li\u003e\n\u003cli\u003eNewcomer BW, Toohey-Kurth K, Zhang Y, Brodersen BW, Marley MS, Joiner KS, Yijing Zhang, Patricia K. Galik, Kay P. Riddell M. Daniel Givens. Laboratory diagnosis and transmissibility of bovine viral diarrhea virus from a bull with a persistent testicular infection. Vet. Microbiol. 2014. 170(3-4):246-57.\u003c/li\u003e\n\u003cli\u003eGard J, Givens M, Stringfellow D. Bovine viral diarrhea virus (BVDV): epidemiologic concerns relative to semen and embryos. Theriogenology. 2007. 68(3):434-42.\u003c/li\u003e\n\u003cli\u003eRikula U, Nuotio L, Laamanen U, Sihvonen L. Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions. Vet. Rec. 2008. 162(3):79-81. \u003c/li\u003e\n\u003cli\u003eOguejiofor CF, Thomas C, Cheng Z, Wathes DC. Mechanisms linking bovine viral diarrhea virus (BVDV) infection with infertility in cattle. Anim. Health Res. Rev. 2019. 20(1):72-85.\u003c/li\u003e\n\u003cli\u003eKirkland P, Mackintosh S, Moyle A. The outcome of widespread use of semen from a bull persistently infected with Pestivirus. Vet. Rec. 1994.135(22):527-9.\u003c/li\u003e\n\u003cli\u003eAitken RJ, Drevet JR. The importance of oxidative stress in determining the functionality of mammalian spermatozoa: a two-edged sword. Antioxid. 2020. 9(2):111. \u003c/li\u003e\n\u003cli\u003eHoma ST, Vassiliou AM, Stone J, Killeen AP, Dawkins A, Xie J, Gould F, Ramsay JWA. A comparison between two assays for measuring seminal oxidative stress and their relationship with sperm DNA fragmentation d semen parameters. Genes. 2019. 10(3):236.\u003c/li\u003e\n\u003cli\u003eAitken RJ, De Iuliis GN, Drevet JR. Role of oxidative stress in the etiology of male infertility and the potential therapeutic value of antioxidants. Oxidants, Antioxidants and Impact of the Oxidative Status in Male Reproduction: Elsevier. 2019. p. 91-100.\u003c/li\u003e\n\u003cli\u003eDabiri M, Talebkhan Garoussi M, Mehrzad J, Tajik P. The Effects of Cytopathic and Non-cytopathic Biotypes of Bovine Viral Diarrhea Virus on Sperm Vitality and Viability of Holstein Dairy Bulls in Vitro. Iran J Vet Med. 2021. 15(2):197-206.\u003c/li\u003e\n\u003cli\u003eVanroose G, Nauwynck H, Soom AV, Vanopdenbosch E, Kruif Ad. Replication of Cytopathic and Noncytopathic Bovine Viral Diarrhea Virus in Zona-Free and Zona-lntact In Vitro-Produced Bovine Embryos and the Effect on Embryo Quality. Biol. Reprod. 1998. 58(3):857-66.\u003c/li\u003e\n\u003cli\u003eVahidi-Ferdowsi P, Mehrzad J, Malvandi A, Hosseinkhani S. Bioluminescence-based detection of astrocytes apoptosis and ATP depletion induced by biologically relevant level aflatoxin B1. World Mycotoxin J. 2018.11(4):589-98.\u003c/li\u003e\n\u003cli\u003eMehrzad J, Duchateau L, Burvenich C. Phagocytic and bactericidal activity of blood and milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows during early lactation. Vet. Microbil. 2009. 134(1-2):106-12.\u003c/li\u003e\n\u003cli\u003eMehrzad J, Malvandi AM, Alipour M, Hosseinkhani S. Environmentally relevant level of aflatoxin B1 elicits toxic pro-inflammatory response in murine CNS-derived cells. Toxicol. Lett. 2017. 279:96-106.\u003c/li\u003e\n\u003cli\u003eMehrzad J, Fazel F, Pouyamehr N, Hosseinkhani S, Dehghani H. Naturally occurring level of aflatoxin B1 injures human, canine and bovine leukocytes through ATP depletion and caspase activation. IJT. 2020. 39(1):30-8. \u003c/li\u003e\n\u003cli\u003eMehrzad J, Hosseinkhani S, Malvandi AM. Human microglial cells undergo proapoptotic induction and inflammatory activation upon in vitro exposure to a naturally occurring level of aflatoxin B1. Neuroimmunomodulation. 2018. 5(3):176-83.\u003c/li\u003e\n\u003cli\u003eBarni F, Lewis SW, Berti A, Miskelly GM, Lago G. Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta. 2007.72(3):896-913.\u003c/li\u003e\n\u003cli\u003eGaroussi MT, Mehrzad J. Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle. Theriogenology. 2011. 75(6):1067-75. \u003c/li\u003e\n\u003cli\u003eMaurer K, Krey T, Moennig V, Thiel H-Jr, Rümenapf T. CD46 is a cellular receptor for bovine viral diarrhea virus. J. Virol. 2004. 78(4):1792-9.\u003c/li\u003e\n\u003cli\u003eGivens MD, Marley MS. Immunology of chronic BVDV infections. Biologicals. 2013.41(1):26-30.\u003c/li\u003e\n\u003cli\u003eDrevet JR, Aitken R. Oxidative damage to sperm DNA: attack and defense. Genetic Damage in Human Spermatozoa: Springer. p. 2019. 107-17.\u003c/li\u003e\n\u003cli\u003eAitken RJ. Impact of oxidative stress on male and female germ cells: implications for fertility. Reprod. 2020. 159(4): R189-R201.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"BVDV, CP, NCP, apoptosis, Oxidative stress, Sperm, In-vitro","lastPublishedDoi":"10.21203/rs.3.rs-7160788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7160788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eBVDV is one of the most infected viral diseases which can impact on genital system of male and female cows. The goal of this study was to see how CP and NCP biotypes of BVDV affected on spermatozoa \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e\u003cp\u003eBVDV-free frozen sperm cells were counted and centrifuged to separate viable sperms. CP and NCP BVDV with 3 different doses of 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e3\u003c/sup\u003e TCID 50/mL were incubated to10\u003csup\u003e5\u003c/sup\u003e sperm/ml for 2 hours. To assess oxidative stress and apoptosis, the luminescence technique was used to quantify ROS, Caspases 7/3 and 9, as well as ATP. They were repeated 3 times.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe effect of virus biotypes on plasma membrane integrity has led to a significant drop in both 10\u003csup\u003e5\u003c/sup\u003e and 10\u003csup\u003e3\u003c/sup\u003e CP and NCP BVDV concentrations (P\u0026thinsp;\u0026le;\u0026thinsp;0.05). The impact of 10\u003csup\u003e5\u003c/sup\u003e dose of CP biotype compared to 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e3\u003c/sup\u003e doses of the same biotype was significant (P \u0026le; 0.05), but no significant difference was observed between the effect of 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e3\u003c/sup\u003e doses. Caspase 3/7 produced showed a significant effect (P \u0026le; 0.05) of both biotypes on sperm cells apoptosis. Caspase 9 produced showed a significant effect (P \u0026le; 0.05) of the three dosages of CP in addition to the high dose of NCP on apoptosis. ATP produced showed a significant effect (P \u0026le; 0.05) of 3 dosages. The effect of 10\u003csup\u003e5\u003c/sup\u003e of CP compared to 10\u003csup\u003e4\u003c/sup\u003e doses and 10\u003csup\u003e4\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e doses were significant (P \u0026le; 0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eWe concluded that BVDV biotypes could affect the vital aspects, dynamic features and also induction of oxidative stress and apoptosis on bovine spermatozoa cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Interaction of CP and NCP BVDV with bovine sperm cells on apoptosis and oxidative stress in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 17:30:47","doi":"10.21203/rs.3.rs-7160788/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-06T15:47:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-15T12:34:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117056140581456045461504326005590794997","date":"2026-02-06T09:07:24+00:00","index":"hide","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-12T04:01:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-30T17:59:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230855456922724935130494353614014780714","date":"2025-11-20T19:57:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13049110439650872877359402953602329012","date":"2025-09-19T18:41:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-17T10:32:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T11:52:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-13T11:59:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-08-13T11:56:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab737b86-7d15-4cef-9d12-ecbb7737a90a","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T15:55:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 17:30:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7160788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7160788","identity":"rs-7160788","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}