بررسی اثرات بایوتایپ های CPو NCP ویروس BVD بر روی وضعیت حیاتی و ماندگاری اسپرم گاوهای نر نژاد هلشتاین در شرائط برون تنی

Introduction

Bovine viral diarrhevirus (BVDV) is one of the most important cattle pathogens that exerts many destructive effects on the health and production of livestock. BVDV is an RNA virus and a member of the genus Pestivirus within the family Flaviviridae (Hopper, 2014). BVDV isolates can be divided into cytopathic (CP) and non-cytopathic (NCP) biotypes, based on their effects on cell culture. Unlike NCP, CP isolates can lead to the formation of vacuoles in cell cytoplasm (cytoplasmic vacuolation) as well as the death of cultured cells (Robert et al., 2004). The NCP biotypes are more prevalent in nature and 60 to 90% of laboratory samples are NCP biotype. Only NCP biotype can cause persistent infection (PI). Epidemiological studies on BVDV in Iran show that it is present in more than 50 to 100% of dairy farms (Garoussi et al., 2019; Rypuła et al., 2020; Foddai et al., 2014). There has been no significant association between the herd size and the prevalence of virus antibody, and animals younger than two years old showed lower serum infection than animals older than two years old (Garoussi et al., 2009). Reproductive diseases following BVDV infection are the most important disorders within the dairy industry. Male and female gonads, as well as all parts of the female reproductive system, can be a suitable medium for BVDV replication (Hopper, 2014). Infected male cows excrete BVDV through semen, but the duration of excretion and its level in persistently infected (PI) animals is much higher than that in cows transiently infected (TI) or acutely infected. Infection of semen with this virus can happen in 4 ways: 1) persistent infection (PI) of bulls during fetal period in uterus, 2) acute infection of bulls after the development of the immune response, 3) chronic testicular infection after prolonged acute infection, and 4) persistent testicular infection from an unknown source (Gard et al., 2007). In laboratory infected bulls, the virus titer in semen varied from 5 to 75 CCID₅₀ (cell culture infective dose 50/mL) and the virus was successfully isolated from raw as well as diluted semen (Kirkland et al., 1991; An et al., 2019). Semen from a PI bull contains high levels of virus (10³ to 10⁷ CCID 50/mL) and even the process of freezing and preparing sperm for artificial insemination does not kill the virus, and subsequent insemination of susceptible heifers with semen from these cows will cause widespread infections in industrial and traditional herds and subsequent damage caused by contamination with both BVDV biotypes. In vitro studies conducted in Iran have been insufficient in this regard.

Given the fact that there are few data regarding the effect of CP and NCP BVDV biotypes on male gametes, this study aimed to investigate the effects of NCP and CP BVDV biotypes on vital status, membrane integrity, and motility of sperm cells from Holstein bulls in vitro.

Materials and Methods

BVDV-Free Sperm Samples

Prior to any experiment, all used sperm samples were tested by PCR with 100% specificity and sensitivity to BVDV in order to be informed of the possible presence of the BVDV (Garoussi and Mehrzad, 2011; Saged Hasan, 2012). All sperm samples were BVDV-free.

Sperm Samples

Frozen BVDV-free sperms of bulls were thawed in a water bath at 37^oC and introduced to the top of a Percoll gradient (45 and 90%; Pharmacia, Uppsala, Sweden). To separate live sperms from dead ones, the sperms were centrifuged for 30 minutes at 2000 × g. The supernatant was removed after centrifugation and the sperm pellet was re-suspended in TALP + BSA, and centrifuged once more for 10 minutes at 750 × g. The resulting sperm pellet was re-suspended to obtain a final concentration of 10⁵ sperm/mL. Frozen sperm samples were obtained from a serial number of the National Breeding Center and Improvement of Animal Production (Karaj-Iran), which were BVDV-free.

Assessment of Sperm Viability

In this study, eosin-nigrosine staining was used to determine whether sperm were alive or dead. To prepare the eosin-nigrosine dye, 1.45 g of sodium citrate was dissolved in 50 mL of deionized water. Five g of nigrosine and 0.835 g of eosin were poured into a suitable container and then sodium citrate solution was added up to 50 mL. The resulting solution was immersed in boiling water for 20 minutes, then filtered and stored at 5^oC in the refrigerator. After preparing the staining solution, 8 drops were mixed with one drop of semen and waited for 3 minutes and the smear was prepared. Sperms which had red heads were considered dead gametes (Agarwal et al., 2016).

Virus

CP and NCP biotypes with high dose (10⁵), medium dose (10⁴), and low dose (10³) (50% tissue culture infectious dose) (TCID 50/mL) were used (Garoussi and Mehrzad, 2011). The virus was cultured in the Minimum Essential Medium (MEM) with fetal calf serum 5% (Vanroose et al., 1998). To observe the cytopathic effects (CPE) of the BVDV, the virus was cultured in Razi bovine kidney (RBK) cells (Razi Vaccine and Serum Research Institute, Karadj-Iran). The virus was then confirmed in both CPE and non-CPE using PCR-specific primers in RBK cells-BVDV co-culture/mixtures. The baseline dose of the virus is usually determined to infect up to 50% of the cells, which is normally done using the virus culture technique in specific cell line. Briefly, the RBK cells were sub-cultured to achieve the desired amount of cells for the RBK cells-BVDV co-culture. These RBK cells were then sub-cultured in the 96-well plates until the cells were adhered and completely covered the surface of the wells (usually it takes about 48 hours for RBK cells). The cells were washed to ensure that only adhered cells are used and non-adhered cells are discarded. The BVDV was then prepared with different dilutions (from 10¹ to 10⁷ in 8 sterile test tubes), and one test tube was normally used as a control. These virus dilutions were mixed and placed adjacent to the RBK cells for about half an hour to allow the virus to enter the cells. It was then washed and incubated with the new culture medium for 3 days. Finally, the cytopathic changes were checked under microscope and the CPE and non-CPE RBK cells in each well were identified (Andrew et al., 2020). The corres-ponding BVDV doses were calculated using the Reed and Muench method. Indeed, different doses of BVDV had different effects on sperm fertilization and adhesion to the egg (Garousi and Mehrzad, 2011; Garoussi et al., 2019).

Membrane Integrity Evaluation

The hypoosmotic swelling (HOS) test was used to assess the sperm membrane integrity. To prepare the HOS solution, 0.9 g of fructose, 0.49 g of sodium citrate, and 0.04 g of citric acid were mixed together and 20 µL of semen were mixed with 200 µL of HOS solution and incubated for 1 hour at 37^oC. The sperm tails that have an integrated and healthy plasma membrane are twisted, and the spermatozoa which have lost the composition of their plasma membranes have a smooth tail (Baiee et al., 2017).

Computer Assisted Semen Analysis

Computer assisted sperm analysis (CASA) is a useful software for the objective evaluation of sperm motility and hence is now frequently used for evaluating semen quality. CASA software (HFT CASA, Hushmand Fanavar, Tehran, Iran) was used to evaluate the effects of two BVDV biotypes on the motility of infected sperm cells. The motility parameters analyzed by CASA system included: total motility (TMOT, %) and progressive motility (PMOT, %), average path velocity (VAP, µm/s), straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), amplitude of lateral head (ALH, µm), mean angular displacement (MAD/D), beat/cross frequency (BCF, Hz), straightness (STR, %), linearity (LIN, %), and wobble (WOB %) as described by Vincent et al. (2018).

Experimental Groups

Treatment

Male gametes were exposed to different doses (high, medium, and low) of CP and NCP BVDV in an incubator at 38.5°C for 2 hours at a concentration of 10⁵ sperm cells/mL. Samples were evaluated for viability features using eosin-nigrosine staining. Live and dead infected sperm cells were assessed. The HOS test was used to assess sperm integrity. CASA system was used to evaluate different motility parameters.

Control

Semen sample with a concentration of 10⁵ sperm/mL without the presence of BVDV biotypes, as well as various treatment groups, were evaluated for viability, membrane integrity, and motility.

All of the above tests for treatment and control groups were repeated 3 times in different time intervals.

Statistical Analysis

The obtained data were analyzed using genera-lized linear model (GLM) in SAS software.

Results

Table 1 shows the effects of BVDV biotypes on sperm viability and plasma membrane integrity in vitro.

It was shown that the number of live sperms in the control group had a range of 72±3.60%, while this value decreased significantly with the increase of virus concentration in both treatment groups (P≤0.05). Moreover, the eff-ect of BVDV biotypes on plasma membrane integrity led to a significant reduction in both high (10⁵) and low (10³) CP and NCP BVDV concentrations (P≤0.05).

Table 2 represents the effects of BVDV biotypes on sperm motility. The results showed a significant difference (P≤0.05) between CP and NCP BVDV biotypes in the control samples and also a significant difference (P≤0.05) between CP and NCP BVDV biotypes in vitro.

Table 1.The effect of CP and NCP BVDV on viability and membrane integrity of sperm cells in vitro

Mean ± SD values in the same rows with different superscripts differ significantly (P≤0.05).

Table 2. CP and NCP BVDV biotypes effect on Holstein bull spermatozoa motility parameters in vitro.

Mean ± SD values in the same rows with different superscripts differ significantly (P≤0.05).

TMOT: Total motility, PMOT: Progressive motility, VCL: Curvilinear velocity, VSL: Straight line velocity, VAP: Average path velocity, MAD (D): Mean angular displacement, ALH: Amplitude of lateral head displacement, BCF: Beat/cross frequency, LIN: Linearity, WOB: Wobble. STR: Straightness.

Discussion