- Email: [email protected]
Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle
Available online at www.sciencedirect.com
Theriogenology 75 (2011) 1067–1075 www.theriojournal.com
Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle M. Talebkhan Garoussia,*, J. Mehrzadb,c a
Section of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad-Iran b Sections Immunology and Biotechnology, Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad-Iran c Biotechnology Research Institute, Ferdowsi University of Mashhad, Mashhad-Iran Received 12 August 2009; received in revised form 10 November 2010; accepted 12 November 2010
Abstract Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (105 and 106/mL) and oocytes were incubated with 106 TCID50/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (105/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 106/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n ⫽ 97), oocytes infected with CP BVDV and incubated at the lower (105/mL) sperm concentration positively affected sperm attachment (n ⫽ 123) to the ZP of fertile oocytes (P ⬍ 0.05). In comparison with the control group (n ⫽ 115), sperm infected with CP BVDV negatively affected sperm binding (n ⫽ 93) to the ZP of infertile oocytes (P ⬍ 0.05). In the second experiment (106 sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n ⫽ 81) to the ZP of fertile oocytes (P ⬍ 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P ⬍ 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF. © 2011 Elsevier Inc. All rights reserved. Keywords: BVDV; Cattle; IVF; Oocytes; Sperm attachment; Zona pellucida
1. Introduction Bovine viral diarrhoea virus (BVDV) is a cattle pathogen affecting multiple body systems, with the
* Corresponding author. Tel.: ⫹98 511 8763851; fax: ⫹98 511 8763852. E-mail address: [email protected] (M.T. Garoussi). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.11.015
reproductive system being one of the most seriously affected [1]. Consequences include abortion, reduced conception rate and persistently infected (PI) calves. It is well known that PI cows infected with non-cytopathogenic (NCP) BVDV frequently give birth to PI calves [2]. The NCP biotype, the most common field isolate, does not induce cell death during replication in cell culture, whereas the cytopathic (CP) biotype, a
1068
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
mutant form of the NCP biotype [3], induces cell necrosis via apoptosis pathway in cell culture [4]. The BVDV is endemic in most countries, although some Scandinavian countries have efficient eradication programs [5]. Using indirect ELISA of bulk milk tanks, the importance of the BVDV has recently been shown in Iran [6]. Exposure of cattle to BVDV may result in either acute, transient (often subclinical), or even PI infections, the latter being initiated by transplacental infection of the developing fetus during early pregnancy (before ⬃125 d of pregnancy) [1]. Although PI calves may survive through birth and develop normally to adulthood, they are immunotolerant to BVDV, and can be persistently or intermittently viraemic without seroconversion [1]. It is well known that PI cows are the primary reservoir for BVDV infection, and thus are a major focus of disease control programs in cattle herds [1]. Based on extensive experimentation, it is clear that the zona pellucida (ZP) is an effective barrier against penetration of some pathogens into the ovum during both in vivo and in vitro fertilization (IVF) [7]. However, some viruses and bacteria can strongly bind to the ZP [8]. Although there are limited data regarding the interaction between infected bovine gametes (sperm and oocytes) and BVDV, it was reported that infected semen used for IVF can carry the virus into the oocyte at fertilization [9]. Furthermore, CD46 acted as a cellular receptor for bovine pestiviruses and was expressed both on sperm and oocytes [10]. In addition, anti-CD46 antibody strongly blocked infection of Madian-Darby Bovine Kidney (MDBK) cells by various BVDV strains [11], confirming concerns of receptor-mediated penetration of BVDV to the host cells. Although BVDV replicates in essentially all organs and tissues, before the occurrence of viremia, the BVDV initially selectively affects tonsils and then spreads to other lymphatic tissues [12]. The interactions of BVDV with CD46 of immune cells, compared to non-immune cells, may induce fundamentally different effects [13]. This virus can also affect and replicate in follicular epithelial cells of PI cattle [14]. Persistently infected bulls shed large quantities of BVDV in semen, in which the virus survives during processing and cryopreservation [15]. Fluids, gametes, and somatic cells from infected cattle are likely to be contaminated with the virus [16]. Using molecular biology techniques, the presence of BVDV has recently been detected in bull semen and bulk tank milk [17,18]. Thus, use of semen or embryos from infected animals can result in spread of BVDV [19]. Considerable effort has been expanded to remove
BVDV from semen of PI bulls [20]. However, BVDV cannot be removed completely from semen in vivo, particularly when the virus is attached to the sperm. Semen used for IVF can carry this virus by sperm and contaminate oocytes at fertilization. Based on available data, it appeared that BVDV can be carried by gametes. Furthermore, it is too difficult to completely remove BVDV from IVF embryos, even with washing done according to International Embryo Transfer Society (IETS) protocols [9]. Fertilization is the consequence of a sequence of events, starting from sperm binding to the ZP of the oocytes, and ending in sperm entry into the cortical ooplasm. A limited number of in vitro experiments have examined whether BVDV infections affect IVF [8,16,17]. However, it remains unclear whether BVDV enhanced or inhibited sperm attachment to the ZP. The objectives of the current study were to determine whether, during IVF: 1) bovine sperm and oocytes incubated with CP and NCP BVDV affected sperm attachment to the ZP; and 2) these BVDV biotypes affected fertilization rate. 2. Material and methods 2.1. Materials free of BVDV and anti-BVDV antibodies Materials from animal sources were bovine serum albumin (BSA), fetal calf serum (FCS), and Hyclone serum. These materials were shown to be free of BVDV by PCR and virus isolation [21], and they were evaluated to be negative to anti-BVDV antibodies by virus neutralization test. 2.2. In vitro oocyte maturation Bovine oocytes were matured using routine techniques [22]. Briefly, compact cumulus-oocyte complexes (COC) were recovered by aspiration of follicles (diameter 2– 6 mm) in abbatoir-derived ovaries. They were subsequently washed four times in a modified Tyrode balanced salt solution, termed HEPES-buffered TALP medium (which consisted of 10 mM HEPES, 0.2 mM sodium pyruvate, 10 mM sodium lactate, 2 mM sodium bicarbonate, 10 g/mL gentamycine sulphate, and 3 mg/mL bovine serum albumin; Sigma-Aldrich, Bornem, Belgium), followed by a further washing with the maturation medium [21]. They were matured in groups of 100 COC in 500 L of maturation medium (no oil overlay) during 24 h of incubation at 38.5 oC in humidified 5% CO2 atmosphere. Maturation medium consisted of tissue culture medium (TCM) 199
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
(Gibco BRL, Merelbeke, Belgium) bicarbonate-buffered medium supplemented with 7.5% Hyclone serum (v/v), 0.5 g/mL follicle-stimulating hormone (FSH), 5 g/mL luteinizing hormone (LH), 0.2 mmol/L sodium pyruvate (Sigma Chemical Co, Bornem, Belgium), 0.4 mmol/L glutamine (Sigma Chemical Co., St. Louis, MO, USA) and 50 g/mL gentamicin sulphate (Gibco BRL). An oocyte with a compact multilayered cumulus investment and homogeneous ooplasm was classified as good quality COC. The oocyte maturation process consists of the completion of nuclear maturation. Therefore, failure of fertilization was characterized by the presence of oocytes at metaphase II. The molecular and structural changes during in vitro maturation (IVM) could support fertilization capacity. Oocytes were denuded of cumulus cells by vortexing in HEPES-buffered TALP medium [21] for 2– 4 min at maximum force (45,000 x g) to remove adherent cumulus cells before being used in IVF. 2.3. Virus The BVDV used in this study were the CP strain Oregon C24V and the NCP isolate No. 22146 [23]. They were cultured in Minimum Essential Medium (MEM) (Sigma-Aldrich, Bornem, Belgium), with 5% Fetal Calf Serum (FCS; N.V. HyClone Europe S. A., Erembodegem, Belgium). 2.4. Sperm Frozen sperm of a BVDV-free bull 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 sperm from dead ones, the sperm were centrifuged for 30 min at 2000 ⫻ g. The supernatant was removed after centrifugation and the sperm pellet was resuspended in TALP ⫹ BSA, and centrifuged once more for 10 min at 750 ⫻ g. The resulting sperm pellet was re-suspended to obtain a final concentration of 107 sperm/mL in IVFTALP with 10 g/mL heparin, and incubated for 1 h at 38 oC. The sperm were then centrifuged again (750 ⫻ g), resuspended in IVF-TALP medium without heparin, and diluted in IVF-TALP to obtain concentrations of 105 and 106 sperm/mL, respectively. 2.5. Experimental design 2.5.1.Control group In vitro fertilization was performed following standard procedures, using 105 or 106 sperm/mL. No BVDV was introduced during the IVF process.
1069
To investigate the effect that incubation of sperm and oocytes with BVDV on sperm attachment to ZP and fertilization rate in IVF, sperm at two concentrations (105 and 106/mL) and cumulus-free and ZP-intact oocytes were allocated into two experiments. 2.5.2.Experiment 1 In this experiment, the sperm concentration for IVF was 105/mL. Male and female gametes were separately incubated with 106 TCID50/mL CP or NCP BVDV for 2 h at 38.5 °C. After incubation, 10 mature cumulus-free intact oocytes were transferred into 50 L of IVF medium containing the infected sperm, whereas 50 L of IVF medium containing 105/mL intact sperm were transferred to 10 mature cumulus-free infected oocytes. 2.5.3.Experiment 2 In the second experiment, the sperm concentration was 106/mL. Sperm and cumulus free ZP-intact oocytes were separately incubated with 106 TCID50/mL of CP or NCP BVDV for 2 h at 38.5 °C. After incubation, 50 L of IVF medium containing 106 infected sperm added to 10 mature cumulus-free intact oocytes. Ten infected oocytes were transferred into 50 L of IVF medium containing 106 intact sperm/mL. To remove non-attached excess sperm, infected oocytes were subsequently washed three times for 10 min in TALP medium in Experiments 1 and 2 [24,25]. In both experiments, approximately 300 oocytes were used, and all assays were in triplicate. In total, 912 oocytes were used. 2.6. Evaluation of number sperm bound to the ZP and the fertilization process The number of sperm attached to the ZP were enumerated separately between fertile and infertile oocytes, which were infected with CP or NCP BVDV in two sperm concentrations; this was in accordance to the method applied in IVF Laboratory, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium [24,25]. To test repeatability, all samples were counted in triplicate. To assess the binding capacity of sperm to the ZP and the rate of fertilization, at 24 h post-incubation, presumptive zygotes were fixed in 2% formalin and 2% glutaraldehyde in phosphate buffered saline (PBS) without calcium and magnesium. Subsequently, zygotes were stained with 10 g/mL Hoechst 33342 (Sigma-Aldrich, Bornem, Belgium) for 10 min. The presumptive zygotes were
1070
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
Fig. 1. Mean ⫾ SEM number of sperm binding to the zona pellucida (ZP) of oocytes, and the effects of bovine male and female gametes infected with CP and NCP BVDV, when a lower sperm concentration (105 sperm/mL) was used in IVF. Upper and lower panels are for fertile and infertile groups, respectively, each with ⬃153 and ⬃285 samples. a,bWithin a panel, categories without a common superscript differed (P ⬍ 0.05).
mounted in 100% glycerol, and blue fluorescence of sperm nuclei and nuclear material of oocytes was evaluated by means of a Leica fluorescence microscope (model DM RBE, Brussels, Belgium). For evaluation of the fertilization process, successful fertilization was characterized by observation of condensation of sperm chromatin and completion of the emission of the second polar body, or by the presence of maternal and paternal pronuclei. Failure of fertilization was characterized by the presence of oocytes at metaphase II. 2.7. Statistical analysis Data for associations between sperm concentration, CP and NCP BVDV infected gametes and fertilization of oocytes, were analyzed with Pearson Chi-Square, using SPSS software version 11.5. For all analyses, P ⬍ 0.05 was considered significant.
3. Results 3.1. Experiment 1 With no contamination of sperm or ova (the control group) and low sperm concentration (105 sperm/mL), an average of 97 and 115 sperm were bound to the ZP of intact fertile and infertile oocytes, respectively (Fig. 1). Similarly, there were analogous results for male and female gametes infected with CP or NCP BVDV on sperm attachment to the ZP of fertile and infertile oocytes in IVF process with lower sperm concentration (105/mL). The sperm bound to the ZP of fertile oocytes of infected bovine male gametes with CP and NCP averaged 110 and 107, respectively. Oocytes contaminated with CP BVDV and incubated with lower sperm concentration had increased sperm attachment (n ⫽ 123) to the ZP of the fertile group (P ⬍ 0.05); this was not detected in infertile oocytes. The number of sperm
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
1071
Fig. 2. Mean ⫾ SEM number of sperm binding to the zona pellucida (ZP) of oocytes, and the effects of bovine male and female gametes infected with CP and NCP BVDV, when a high sperm concentration (106 sperm/mL) was used in IVF. Because sperm binding to the ZP in control groups of fertile and infertile oocytes was very high (and therefore not countable), the control groups are not shown. Upper and lower panels are for fertile and infertile groups, respectively, each with ⬃148 and ⬃326 samples, respectively. a-cWithin a panel, categories without a common superscript differed (P ⬍ 0.05).
attached to the ZP of fertile oocytes contaminated with NCP BVDV was 113. Sperm contaminated with CP BVDV had reduced sperm binding (n ⫽ 93) to the ZP of infertile oocytes (P ⬍ 0.05). There were no significant differences among the infected sperm with NCP BVDV (n ⫽ 113), oocytes with CP (n ⫽ 120) and NCP (n ⫽ 124) BVDV in comparison with control gametes (n ⫽ 97; P ⬎ 0.05). 3.2. Experiment 2 For the high level of sperm concentration (106 sperm/mL), the effects of infected male and female gametes with CP and NCP BVDV on sperm binding to the ZP of fertile and infertile oocytes in the IVF process are shown (Fig. 2). Sperm attachment to control group fertile and infertile oocytes was very high and therefore
uncountable (no data to show). However, with the higher sperm concentration, only infected sperm with CP BVDV had reduced binding capacity (n ⫽ 81) to the ZP of fertile oocytes in comparison with infected sperm with NCP BVDV (n ⫽ 125) of fertile group. Nevertheless, the negative effect was also noticeable on sperm attachment capacity (n ⫽ 127) to the ZP of the fertile oocytes infected with CP BVDV biotype, in comparison with infected fertile oocytes with NCP BVDV (n ⫽ 151). There were no significant differences between sperm attachment to the ZP of infertile oocytes of infected male gametes with CP (n ⫽ 104) or NCP (n ⫽ 122) BVDV, as was also true in infertile female gametes infected with CP (n ⫽ 204) or NCP (n ⫽ 195) BVDV. Effects on fertilization rate of the presence of CP
1072
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
Table 1 Rate of successful in vitro fertilization in bovine oocytes incubated with CP and NCP BVDV of sperm combined with oocytes incubated with 106 TCID50/mL of CP or NCP BVDV at two sperm concentrations. Sperm/mL 105 106 a-c
Rate of IVF (%) in control and treatment groups Control
Sperm and CP
Sperm and NCP
Oocytes and CP
Oocytes and NCP
48%a (43/89) 74%a (64/86)
35%a (30/84) 4%c (4/99)
29%b (23/80) 14%c (13/91)
38%a (37/96) 19%c (19/99)
22%b (20/89) 48%c (48/99)
Within a row, percentages without a common superscript differed (P ⬍ 0.05).
and NCP BVDV on sperm and oocytes during the IVF process are shown (Table 1). Compared to the control group, fertilization rates were significantly reduced both in the low (105 sperm/mL) and high (106 sperm/ mL) sperm concentration groups with male and female gametes infected with NCP BVDV biotypes (P ⬍ 0.05). However, fertilization rate did not differ significantly with the low sperm concentration in gametes infected with CP BVDV. However, fertilization rate in infected NCP BVDV sperm (29%) and oocytes (22%) differed (P ⬍ 0.05). Furthermore, fertilization rates were reduced from sperm and oocytes infected with CP BVDV at the higher sperm concentration (P ⬍ 0.05). 4. Discussion In this study, the only treatment that increased sperm binding to the ZP was oocytes infected with CP BVDV at the lower sperm concentration (105sperm/mL). With the higher sperm concentration (106 sperm/mL), the number of sperm binding to the ZP of control treatment oocytes was so high as to be uncountable. In the group of fertilized oocytes, sperm attachment markedly decreased when sperm were infected with CP BVDV in comparison with NCP BVDV. The underlying reasons for the differences in responses of sperm and ova to the CP and NCP BVDV biotypes were unclear. There is little detailed knowledge of the molecular mechanisms responsible for the entry of BVDV biotypes into bovine cells, but several reasons can be postulated for observed differences. Some glycoproteins (gp), especially gp48 and gp53, present in the viral envelope are thought to be involved in attachment of the virus to its host cells [26,27]. Furthermore, although different BVDV biotypes do not necessarily have the same receptor, the presence of multiple receptors for BVDV attachment to the cell surface may contribute to the BVDV attachment to the host cells. It has also been reported that BVDV uses more than one receptor for binding [28]. Some viruses can attach strongly to oocytes [8]; for example, there has recently been better understanding regarding the
mechanism by which Pestiviruses attach to their host cells. Bovine CD46 acts as a cellular receptor for BVDV [29]. Perhaps CP and NCP BVDV use different receptors for cell attachment or entry; further basic investigations are needed to confirm this theory. In the current study, the presence of CP and NCP BVDV during the IVF process with two different sperm concentrations significantly reduced the fertilization rate. The fertilization rate of control group oocytes at the lower sperm concentration was lower than at the higher sperm concentration; this lower fertilization rate could have been due to the 10-fold lower concentration of sperm. Conversely, at the concentration of 105 sperm/mL, fertilization rate of CP BVDV-infected oocytes was not affected. In a previous study, fertilization rate of intact oocytes was reduced only with sperm that had been incubated with CP BVDV [30]. Influences of CP and NCP BVDV in decreasing fertilization rate during IVF were manifested mainly by reduced sperm attachment to the fertile and infertile oocytes at the higher sperm concentration. Therefore, in the control group, sperm attachment to the ZP of oocytes was too high to be counted, due to normal binding of the sperm to the ZP; this further confirmed the negative effects of BVDV on the sperm-oocyte interaction. However, with lower sperm concentration, only fertile oocytes infected with CP BVDV differed in sperm binding to the ZP. Although, it has been shown that the intact ZP of oocytes can act as a penetration barrier against pathogens [31], in oocytes infected with BVDV, this barrier may have been compromised, facilitating sperm penetration during fertilization [32]. The origin of oocytes is of considerable importance in the sanitary risks associated with production of in vitro embryos. Oocytes are surrounded by cumulus cells. The role of the surrounding cumulus cells in protecting IVF embryo from contamination and chemical damage is very important [33,34]. Semen is rarely considered a source of virus transmission [35]. However, viruses can easily bind to the male gamete [20,25,36]. Pathogens such as BVDV can contaminate follicular fluid, granolosa, and cumulus cells, and even
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
cells from the ovarian stroma [37]. Therefore, BVDV biotypes can bind to the ZP and penetrate into the oocytes during fertilization. Most studies reported lower rates of fertilization and embryo development during BVDV infection [38,39], which may depend on BVDV strains, biotypes [21], and the dose of the virus [39]. Inhibition of sperm attachment to the ZP may be due to a toxic or cytopathic influence of BVDV. Perhaps BVDV reduces the ability of sperm to attach to the ZP by decreasing sperm motility (total and progressive), sperm capacitation and the acrosome reaction. The effect of BVDV infection at the time of IVF has been the focus of several experimental studies [40,41]. In France, fertilization and embryo cleavage rates were reduced when semen from a PI bull was used for IVF [40]. In a subsequent study, there were three groups of in vitro-matured oocytes: the first group was exposed to semen from a PI bull (positive control); the second was exposed to the same semen after it had been treated during the ‘swim-up’ phase with a specific anti-BVDV immunoglobulin, and the third group was exposed to semen from a BVDV-free bull (negative control) [41]. Proportions of oocytes developing to the blastocyst in the three groups were 4.0, 8.2, and 13.9%, respectively. The authors suggested that the low rate of embryo development in the first group was a specific effect of the virus. They also claimed that the marginally reduced blastocyst-formation rate in the second group, in which the virus neutralized, may have been due to poor semen quality in the PI bull. The present study confirmed the negative effects of CP and NCP BVDV on sperm attachment on the ZP and fertilization rates. This effect could result mainly from the reduction of sperm attachment to the ZP of oocytes [30]. In a study by Bielanski and Loewen, when a NCP BVDV strain (105 TCID50/mL) was incubated with semen from an uninfected bull prior to IVF, 22% of embryos developed to blastocysts. Moreover, the percentages of oocytes which cleaved and the percentages of embryos which developed to blastocysts were 47, 51, and 20%, and 17, 20, and 13% using semen from the three PI bulls, respectively, and were 53 and 22% using pooled semen from three uninfected bulls [41]. There are considerable data regarding the interaction of BVDV with in vitro-produced embryos, but few data regarding whether semen used for IVF can lead to transfer of BVDV, via sperm, to oocytes during fertilization [30]. The BVDV replicates entirely within the cytoplasm of the host cells. High virus concentrations have been reported in the blood, nasal secretions, sa-
1073
liva, tears, milk, urine, feces, lymph nodes, and reproductive organs of PI cattle. Tissue distribution of BVDV is widespread in clinically infected cattle [42,43,44]. It has also been shown that BVDV infection can be transmitted through the semen of transiently infected bulls under field conditions [45]. The BVDV can be the cause of infertility in the field due to early embryo death, reduction of sperm attachment to the ZP, interference with cells in the reproductive tract (e.g., cumulus and granulosa cells) and follicular fluid performance [46]. Cattle herds can be infected by BVDV. Recently, it has been shown that dairy cattle in Iran were infected with this virus [6,47], which can be an underlying cause of infertility and reproductive problems in dairy herds. In conclusion, both CP BVDV and NCP BVDV interacted with sperm and oocyte surfaces in cattle; this interaction influenced sperm binding to the ZP of oocytes and IVF rate. Oocytes infected with CP or NCP BVDV compromised sperm attachment to the ZP. At the higher level of sperm concentration evaluated in this study, both biotypes of BVDV had greater reduction in sperm attachment to the ZP than at the lower sperm concentration. Regardless, the exact mechanisms of BVDV attachment on male and female gametes need more fundamental investigations. Acknowledgments This study was supported financially by Ferdowsi University of Mashhad, Mashhad-Iran and the Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. The authors gratefully thank Professor Ann Van Soom, Professor Hans Nauwynck, and Professor Aart de Kruif for their support. The virus stock was kindly provided by the Veterinary and Agriculture Research Center, Brussels, Belgium. References [1] Radostits OM, Gay CC. Hinchcliff KW. Constable PD. Veterinary Medicine. 10th Edition, W. B. Saunders, England. 2007, p. 1249 – 60. [2] McClurkin AW, Littledike ET, Cutlip RC, Frank GH, Ceria MF, Bolin SR. Production of cattle immunotolerant to bovine viral diarrhea virus. Can J Comp Med 1984;48:156 – 61. [3] Donis RO. Molecular biology of bovine viral diarrhea virus and its interactions with the host. Vet Clin North Am Food Anim Prac 1995;11:393– 423. [4] Zhang G, Aldridge S, Clarke MC, McCauley JW. Cell death induced by cytopathic bovine viral diarrhoea virus is mediated by apoptosis. J Gen Virol 1996;77:1677– 81.
1074
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
[5] Lindberg A. The Nordic bovine viral diarrhoea virus eradication programmes—their success and future. Cattle Pract 2004;12: 3–5. [6] Talebkhan Garoussi M, Haghparast A, Estajee H. Prevalence of bovine viral diarrhoea virus antibodies in bulk tank milk of industrial dairy cattle herds in suburb of Mashhad-Iran. Prev Vet Med 2008;84:171– 6. [7] Stringfellow D, Wrathall A. Epidemiological implications of the production and transfer of IVF embryos. Theriogenology 1995;43:89 –96. [8] Bielanski A. A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and related new reproductive technologies. Biotech Adv 1997;15: 633–56. [9] Wrathall AE, Simmons HA, Van Soom A. Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen. Theriogenology 2006;65:247–74. [10] Iqbal M, McCauley JW. Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis. J Gen Virol 2002;83: 2153–9. [11] Krey T, Himmelrreich A, Heimann M, Menge C, Jurgen H, Maurer K, Rumenapf T. Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1. J Virol 2006;80:3912–22. [12] Liebler TEM, Greiser W, Pohlenz JF. Organ and tissue distribution of the antigen of the cytopathogenic bovine virus diarrhea virus in the early and advanced phase of experimental mucosal disease. Arch Virol 1997;142:1613–34. [13] Cattaneo R. Four viruses, two bacteria and one receptor: membrane cofactor protein (CD46) as pathogens magnet. J Virol 2004;78:4385– 8. [14] Tsuboi T, Imada T. Bovine viral diarrhea virus replication in bovine follicular epithelial cells derived from persistently infected heifers. J Vet Med Sci 1998;60:569 –72. [15] Kirkland PD, Mackintosh SG, Moyle A. The outcome of wide spread use of semen from a bull persistently infected with pestivirus. Vet Rec. 1994;135:527–9. [16] Gard JA, Given MD, Stringfellow DA. Bovine diarrhea virus (BVDV): Epidemiologic concerns relative to semen and embryos. Theriogenology 2007;68:434 – 42. [17] Given MD, Heath AM, Brock KV, Brodersen BW, Carson RL, Stringfellow DA. Detection of bovine viral diarrhea virus in semen obtained after inoculation of seronegative postbubertal bulls. Am J Vet Res 2003;64:428 –34. [18] Talebkhan Garoussi M, Bassami MR, Afshari SE. Detection of bovine viral diarrhea virus using a nested RT-PCR assay in bulk milk samples of dairy cattle herds in suburb of Mashhad-Iran. Iran J Biotechnol 2007;5:52–5. [19] Gard JA, Given MD, Stringfellow DA. Bovine diarrhea virus (BVDV): Epidemiologic concerns relative to semen and embryos. Theriogenology 2007;68:434 – 42. [20] Bielanski A, Dubuc C, Hare WCD. Failure to remove bovine viral diarrhea virus from bull semen by swim-up and other separatory sperm techniques associated with in vitro fertilization. Reprod Dom Anim 1992;27:303– 6. [21] Van Soom A, Vanopdenbosh E, Mahmouzadeh A R, de Kruif A. In vitro production of Belgian Blue cattle embryo: some data on the risk of viral infections. Vlaams Diergenees Kundig Tijdschrift 1994;63:139 – 45.
[22] Mahmoudzadeh AR, Van Soom A, Bols P, Ysebaert M T, de Kruif A. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of development stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival. J Reprod Fertil 1995;103:33–9. [23] Vanroose G, Nauwynck H, Van Soom A, Vanopdenbosch E, de kruif A. Replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona free and zona-intact in vitro produced bovine embryos and the effect on embryo quality. Biol Reprod 1998;58:857– 66. [24] Vanroose G. Interaction of bovine herpesvirus-1 and bovine viral diarrhea virus with bovine gametes and in-vitro produced embryos. Ghent University. Ph. D thesis. 1999;174-5. [25] Tanghe S, Vanroose G, Van Soom A, Duchateau L, Ysebaert MT, Kerkhofs P, Thiry E, Van Drunen Little-van den Hurk S, Van Oostveldt P, Nauwynck H. Inhibition of bovine–zona binding by bovine herpesvirus-1. Reproduction 2005;130:251–9. [26] Donis RO, Dubovi EJ. Glycoproteins of bovine viral diarrhea– mucosal disease virus in infected bovine cells. J Gen Virol 1987;68:1607–16. [27] Boulanger D, Waxweiler S, Karelle L, Loncar M, Mignon B, Dubuisson Thiry E, Pastoret PP. Characterization of monoclonal antibodies to bovine viral diarrhea virus: evidence of a neutralization activity against gp48 in the presence of antimouse immunoglobulin serum. J Gen Virol 1991;72:1389 – 402. [28] Schelp C, Greiser-Wilke I, Wolf G, Beer M, Moennig V, Liess B. Identification of cell member proteins linked to susceptibility to bovine viral diarrhea virus infection. Arch Virol 1995;140: 1997–2009. [29] Maurer KT, Krey V, Moennig H, Thiel J, Rumenapf T. CD46 is a cellular receptor for bovine viral diarrhea virus. J Virol 2004; 78:1792–9. [30] Talebkhan Garoussi M. The effects of cytopathic and noncytopathic Bovine Viral Diarrhoea Virus with sperm cells on in vitro fertilization of bovine oocytes. Vet Res Commun 2007;31:365– 70. [31] Vanroose G, Nauwynck H, Van Soom A, Ysebaert MT, Charlier G, Van Oostveldt P. Structural aspects of the zona pellucida of in-vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biol Reprod 2000; 62:463–9. [32] Le Tallec B, Ponsart C, Marquant-Le Guienne B, Guérin B. Risks of transmissible diseases in relation to embryo transfer. Reprod Nutr Develop 2001;41:439 –50. [33] Stringfellow DA, Seidel S. Manual of the International Embryo Transfer Society. IETS, Urbana, Illinois. 1998. [34] Tanghe S, Van Soom A, Mehrzad J, Maes D, Duchateau L, de Kruif A. Cumulus contributions during bovine fertilization in vitro. Theriogenology 2003;60:135– 49. [35] Bielanski A. The problems and risks of the contamination of germplasm with pathogenic microbes using current reproductive technologies. In: Enne V, Greppi E, Lauria A, editors. Reproduction and Animal Breeding: Advances and Strategy. Elsevier, 1995, p. 99 –116. [36] Tsuboi T, Osawa T, Kimura K, Kubo M, Haritani M. Experimental infection of early pregnant cows with bovine viral diarrhea virus: Transmission of virus to the reproductive tract and conceptus. Res Vet Sci 2010 (In press). [37] Booth P J, Stevens DA, Collins ME, Brownlie J. Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle. J Reprod Fertil 1995;105:17–24.
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075 [38] Guérin B, Chaffaux S, Marquant-Leguienne B, Allietta M, Thibier M. IVF and IV culture of bovine embryos using semen from a bull persistently infected with BVD. Theriogenology 1992;37:217. [39] Vanroose G, Nauwynck H, Van Soom A, Vanopdenbosch E, de Kruif A. Effect of bovine herpesvirus-1 or bovine viral diarrhea virus on development of in vitro-produced bovine embryos. Mol Reprod Dev 1999;54:255– 63. [40] Allietta M, Guerin B, Marquant-Le GB, Thibier M. The effect of neutralization of BVD/MD virus present in bovine semen on the IVF and development of bovine embryos. Theriogenology 1995;43:156. [41] Bielanski A, Loewen K. In vitro fertilization of bovine oocytes with semen from bulls persistently infected with bovine viral diarrhoea virus. Anim Reprod Sci 1994;35:183–9. [42] Avery B, Greve T, Ronsholt L, Botner A. Virus screening of a bovine in vitro embryo production system. Vet Rec 1993;132: 660.
1075
[43] Baszler TV, Evermann JF, Kaylor PS, Byington TC, Dilbeck PM. Diagnosis of naturally occurring bovine viral diarrhea virus infections in ruminants using monoclonal antibody-based immunohistochemistry. Vet Pathol 1995;32:609 –18. [44] Brownlie J. The pathways for bovine virus diarrhoea virus biotypes in the pathogenesis of disease. Arch Virol 1991;3:79 – 96. [45] Rikula U, Nuotio L, Laamanen UI, Sihvonen L. Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions. Vet Rec 2008; 162:79 – 81. [46] Daniel LG. Reproductive consequences of infection with Bovine Viral Diarrhea Virus. Vet Clin Food Anim 2004;20: 5–19. [47] Talebkhan Garoussi M, Haghparast A, Hajenejad MR. Prevalence of Bovine Viral Diarrhoea Virus antibodies among the industrial dairy cattle herds in suburb of Mashhad-Iran. Trop Anim Health Prod 2009;41:663–7.
Theriogenology 75 (2011) 1067–1075 www.theriojournal.com
Effect of bovine viral diarrhoea virus biotypes on adherence of sperm to oocytes during in-vitro fertilization in cattle M. Talebkhan Garoussia,*, J. Mehrzadb,c a
Section of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad-Iran b Sections Immunology and Biotechnology, Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad-Iran c Biotechnology Research Institute, Ferdowsi University of Mashhad, Mashhad-Iran Received 12 August 2009; received in revised form 10 November 2010; accepted 12 November 2010
Abstract Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is one of the most important pathogens of dairy cattle; it can cause several clinical syndromes, ranging from subclinical to severe disease. The objectives of the current studies were to assess the effects of two biotypes of BVDV on sperm attachment to the zona pellucida (ZP) of oocytes and on fertilization rate in bovine in vitro fertilization (IVF). In two experiments, sperm at two concentrations (105 and 106/mL) and oocytes were incubated with 106 TCID50/mL cythopatic (CP) or noncythopatic (NCP) BVDV. In the first experiment, with the lower sperm concentration (105/mL), male and female gametes were infected with CP or NCP BVDV, whereas in the second experiment, the sperm concentration was 106/mL, and sperm and oocytes were also infected with CP or NCP BVDV. The number of sperm attached to the ZP and the fertilization rate were evaluated with fluorescence microscopy on the ZP of fertile and infertile oocytes. In the first experiment, compared to the control group (n ⫽ 97), oocytes infected with CP BVDV and incubated at the lower (105/mL) sperm concentration positively affected sperm attachment (n ⫽ 123) to the ZP of fertile oocytes (P ⬍ 0.05). In comparison with the control group (n ⫽ 115), sperm infected with CP BVDV negatively affected sperm binding (n ⫽ 93) to the ZP of infertile oocytes (P ⬍ 0.05). In the second experiment (106 sperm/mL), for both fertile and infertile oocyte groups, sperm attachment in the control group was very high and deemed uncountable. However, in treated groups, the number of sperm attached to the ZP was countable. Only sperm infected with CP BVDV negatively affected sperm binding capacity (n ⫽ 81) to the ZP of fertile oocytes (P ⬍ 0.05). Although CP and NCP BVDV significantly reduced the fertilization rate of oocytes incubated with a higher sperm concentration, with the lower sperm concentration, only NCP BVDV significantly diminished fertilization rate with contaminated sperm and oocytes (P ⬍ 0.05). In conclusion, this study supported the detrimental impacts of sperm or ooctyes infected with CP or NCP BVDV on sperm attachment to the ZP of bovine oocytes and on fertilization rate during bovine IVF. © 2011 Elsevier Inc. All rights reserved. Keywords: BVDV; Cattle; IVF; Oocytes; Sperm attachment; Zona pellucida
1. Introduction Bovine viral diarrhoea virus (BVDV) is a cattle pathogen affecting multiple body systems, with the
* Corresponding author. Tel.: ⫹98 511 8763851; fax: ⫹98 511 8763852. E-mail address: [email protected] (M.T. Garoussi). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.11.015
reproductive system being one of the most seriously affected [1]. Consequences include abortion, reduced conception rate and persistently infected (PI) calves. It is well known that PI cows infected with non-cytopathogenic (NCP) BVDV frequently give birth to PI calves [2]. The NCP biotype, the most common field isolate, does not induce cell death during replication in cell culture, whereas the cytopathic (CP) biotype, a
1068
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
mutant form of the NCP biotype [3], induces cell necrosis via apoptosis pathway in cell culture [4]. The BVDV is endemic in most countries, although some Scandinavian countries have efficient eradication programs [5]. Using indirect ELISA of bulk milk tanks, the importance of the BVDV has recently been shown in Iran [6]. Exposure of cattle to BVDV may result in either acute, transient (often subclinical), or even PI infections, the latter being initiated by transplacental infection of the developing fetus during early pregnancy (before ⬃125 d of pregnancy) [1]. Although PI calves may survive through birth and develop normally to adulthood, they are immunotolerant to BVDV, and can be persistently or intermittently viraemic without seroconversion [1]. It is well known that PI cows are the primary reservoir for BVDV infection, and thus are a major focus of disease control programs in cattle herds [1]. Based on extensive experimentation, it is clear that the zona pellucida (ZP) is an effective barrier against penetration of some pathogens into the ovum during both in vivo and in vitro fertilization (IVF) [7]. However, some viruses and bacteria can strongly bind to the ZP [8]. Although there are limited data regarding the interaction between infected bovine gametes (sperm and oocytes) and BVDV, it was reported that infected semen used for IVF can carry the virus into the oocyte at fertilization [9]. Furthermore, CD46 acted as a cellular receptor for bovine pestiviruses and was expressed both on sperm and oocytes [10]. In addition, anti-CD46 antibody strongly blocked infection of Madian-Darby Bovine Kidney (MDBK) cells by various BVDV strains [11], confirming concerns of receptor-mediated penetration of BVDV to the host cells. Although BVDV replicates in essentially all organs and tissues, before the occurrence of viremia, the BVDV initially selectively affects tonsils and then spreads to other lymphatic tissues [12]. The interactions of BVDV with CD46 of immune cells, compared to non-immune cells, may induce fundamentally different effects [13]. This virus can also affect and replicate in follicular epithelial cells of PI cattle [14]. Persistently infected bulls shed large quantities of BVDV in semen, in which the virus survives during processing and cryopreservation [15]. Fluids, gametes, and somatic cells from infected cattle are likely to be contaminated with the virus [16]. Using molecular biology techniques, the presence of BVDV has recently been detected in bull semen and bulk tank milk [17,18]. Thus, use of semen or embryos from infected animals can result in spread of BVDV [19]. Considerable effort has been expanded to remove
BVDV from semen of PI bulls [20]. However, BVDV cannot be removed completely from semen in vivo, particularly when the virus is attached to the sperm. Semen used for IVF can carry this virus by sperm and contaminate oocytes at fertilization. Based on available data, it appeared that BVDV can be carried by gametes. Furthermore, it is too difficult to completely remove BVDV from IVF embryos, even with washing done according to International Embryo Transfer Society (IETS) protocols [9]. Fertilization is the consequence of a sequence of events, starting from sperm binding to the ZP of the oocytes, and ending in sperm entry into the cortical ooplasm. A limited number of in vitro experiments have examined whether BVDV infections affect IVF [8,16,17]. However, it remains unclear whether BVDV enhanced or inhibited sperm attachment to the ZP. The objectives of the current study were to determine whether, during IVF: 1) bovine sperm and oocytes incubated with CP and NCP BVDV affected sperm attachment to the ZP; and 2) these BVDV biotypes affected fertilization rate. 2. Material and methods 2.1. Materials free of BVDV and anti-BVDV antibodies Materials from animal sources were bovine serum albumin (BSA), fetal calf serum (FCS), and Hyclone serum. These materials were shown to be free of BVDV by PCR and virus isolation [21], and they were evaluated to be negative to anti-BVDV antibodies by virus neutralization test. 2.2. In vitro oocyte maturation Bovine oocytes were matured using routine techniques [22]. Briefly, compact cumulus-oocyte complexes (COC) were recovered by aspiration of follicles (diameter 2– 6 mm) in abbatoir-derived ovaries. They were subsequently washed four times in a modified Tyrode balanced salt solution, termed HEPES-buffered TALP medium (which consisted of 10 mM HEPES, 0.2 mM sodium pyruvate, 10 mM sodium lactate, 2 mM sodium bicarbonate, 10 g/mL gentamycine sulphate, and 3 mg/mL bovine serum albumin; Sigma-Aldrich, Bornem, Belgium), followed by a further washing with the maturation medium [21]. They were matured in groups of 100 COC in 500 L of maturation medium (no oil overlay) during 24 h of incubation at 38.5 oC in humidified 5% CO2 atmosphere. Maturation medium consisted of tissue culture medium (TCM) 199
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
(Gibco BRL, Merelbeke, Belgium) bicarbonate-buffered medium supplemented with 7.5% Hyclone serum (v/v), 0.5 g/mL follicle-stimulating hormone (FSH), 5 g/mL luteinizing hormone (LH), 0.2 mmol/L sodium pyruvate (Sigma Chemical Co, Bornem, Belgium), 0.4 mmol/L glutamine (Sigma Chemical Co., St. Louis, MO, USA) and 50 g/mL gentamicin sulphate (Gibco BRL). An oocyte with a compact multilayered cumulus investment and homogeneous ooplasm was classified as good quality COC. The oocyte maturation process consists of the completion of nuclear maturation. Therefore, failure of fertilization was characterized by the presence of oocytes at metaphase II. The molecular and structural changes during in vitro maturation (IVM) could support fertilization capacity. Oocytes were denuded of cumulus cells by vortexing in HEPES-buffered TALP medium [21] for 2– 4 min at maximum force (45,000 x g) to remove adherent cumulus cells before being used in IVF. 2.3. Virus The BVDV used in this study were the CP strain Oregon C24V and the NCP isolate No. 22146 [23]. They were cultured in Minimum Essential Medium (MEM) (Sigma-Aldrich, Bornem, Belgium), with 5% Fetal Calf Serum (FCS; N.V. HyClone Europe S. A., Erembodegem, Belgium). 2.4. Sperm Frozen sperm of a BVDV-free bull 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 sperm from dead ones, the sperm were centrifuged for 30 min at 2000 ⫻ g. The supernatant was removed after centrifugation and the sperm pellet was resuspended in TALP ⫹ BSA, and centrifuged once more for 10 min at 750 ⫻ g. The resulting sperm pellet was re-suspended to obtain a final concentration of 107 sperm/mL in IVFTALP with 10 g/mL heparin, and incubated for 1 h at 38 oC. The sperm were then centrifuged again (750 ⫻ g), resuspended in IVF-TALP medium without heparin, and diluted in IVF-TALP to obtain concentrations of 105 and 106 sperm/mL, respectively. 2.5. Experimental design 2.5.1.Control group In vitro fertilization was performed following standard procedures, using 105 or 106 sperm/mL. No BVDV was introduced during the IVF process.
1069
To investigate the effect that incubation of sperm and oocytes with BVDV on sperm attachment to ZP and fertilization rate in IVF, sperm at two concentrations (105 and 106/mL) and cumulus-free and ZP-intact oocytes were allocated into two experiments. 2.5.2.Experiment 1 In this experiment, the sperm concentration for IVF was 105/mL. Male and female gametes were separately incubated with 106 TCID50/mL CP or NCP BVDV for 2 h at 38.5 °C. After incubation, 10 mature cumulus-free intact oocytes were transferred into 50 L of IVF medium containing the infected sperm, whereas 50 L of IVF medium containing 105/mL intact sperm were transferred to 10 mature cumulus-free infected oocytes. 2.5.3.Experiment 2 In the second experiment, the sperm concentration was 106/mL. Sperm and cumulus free ZP-intact oocytes were separately incubated with 106 TCID50/mL of CP or NCP BVDV for 2 h at 38.5 °C. After incubation, 50 L of IVF medium containing 106 infected sperm added to 10 mature cumulus-free intact oocytes. Ten infected oocytes were transferred into 50 L of IVF medium containing 106 intact sperm/mL. To remove non-attached excess sperm, infected oocytes were subsequently washed three times for 10 min in TALP medium in Experiments 1 and 2 [24,25]. In both experiments, approximately 300 oocytes were used, and all assays were in triplicate. In total, 912 oocytes were used. 2.6. Evaluation of number sperm bound to the ZP and the fertilization process The number of sperm attached to the ZP were enumerated separately between fertile and infertile oocytes, which were infected with CP or NCP BVDV in two sperm concentrations; this was in accordance to the method applied in IVF Laboratory, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium [24,25]. To test repeatability, all samples were counted in triplicate. To assess the binding capacity of sperm to the ZP and the rate of fertilization, at 24 h post-incubation, presumptive zygotes were fixed in 2% formalin and 2% glutaraldehyde in phosphate buffered saline (PBS) without calcium and magnesium. Subsequently, zygotes were stained with 10 g/mL Hoechst 33342 (Sigma-Aldrich, Bornem, Belgium) for 10 min. The presumptive zygotes were
1070
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
Fig. 1. Mean ⫾ SEM number of sperm binding to the zona pellucida (ZP) of oocytes, and the effects of bovine male and female gametes infected with CP and NCP BVDV, when a lower sperm concentration (105 sperm/mL) was used in IVF. Upper and lower panels are for fertile and infertile groups, respectively, each with ⬃153 and ⬃285 samples. a,bWithin a panel, categories without a common superscript differed (P ⬍ 0.05).
mounted in 100% glycerol, and blue fluorescence of sperm nuclei and nuclear material of oocytes was evaluated by means of a Leica fluorescence microscope (model DM RBE, Brussels, Belgium). For evaluation of the fertilization process, successful fertilization was characterized by observation of condensation of sperm chromatin and completion of the emission of the second polar body, or by the presence of maternal and paternal pronuclei. Failure of fertilization was characterized by the presence of oocytes at metaphase II. 2.7. Statistical analysis Data for associations between sperm concentration, CP and NCP BVDV infected gametes and fertilization of oocytes, were analyzed with Pearson Chi-Square, using SPSS software version 11.5. For all analyses, P ⬍ 0.05 was considered significant.
3. Results 3.1. Experiment 1 With no contamination of sperm or ova (the control group) and low sperm concentration (105 sperm/mL), an average of 97 and 115 sperm were bound to the ZP of intact fertile and infertile oocytes, respectively (Fig. 1). Similarly, there were analogous results for male and female gametes infected with CP or NCP BVDV on sperm attachment to the ZP of fertile and infertile oocytes in IVF process with lower sperm concentration (105/mL). The sperm bound to the ZP of fertile oocytes of infected bovine male gametes with CP and NCP averaged 110 and 107, respectively. Oocytes contaminated with CP BVDV and incubated with lower sperm concentration had increased sperm attachment (n ⫽ 123) to the ZP of the fertile group (P ⬍ 0.05); this was not detected in infertile oocytes. The number of sperm
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
1071
Fig. 2. Mean ⫾ SEM number of sperm binding to the zona pellucida (ZP) of oocytes, and the effects of bovine male and female gametes infected with CP and NCP BVDV, when a high sperm concentration (106 sperm/mL) was used in IVF. Because sperm binding to the ZP in control groups of fertile and infertile oocytes was very high (and therefore not countable), the control groups are not shown. Upper and lower panels are for fertile and infertile groups, respectively, each with ⬃148 and ⬃326 samples, respectively. a-cWithin a panel, categories without a common superscript differed (P ⬍ 0.05).
attached to the ZP of fertile oocytes contaminated with NCP BVDV was 113. Sperm contaminated with CP BVDV had reduced sperm binding (n ⫽ 93) to the ZP of infertile oocytes (P ⬍ 0.05). There were no significant differences among the infected sperm with NCP BVDV (n ⫽ 113), oocytes with CP (n ⫽ 120) and NCP (n ⫽ 124) BVDV in comparison with control gametes (n ⫽ 97; P ⬎ 0.05). 3.2. Experiment 2 For the high level of sperm concentration (106 sperm/mL), the effects of infected male and female gametes with CP and NCP BVDV on sperm binding to the ZP of fertile and infertile oocytes in the IVF process are shown (Fig. 2). Sperm attachment to control group fertile and infertile oocytes was very high and therefore
uncountable (no data to show). However, with the higher sperm concentration, only infected sperm with CP BVDV had reduced binding capacity (n ⫽ 81) to the ZP of fertile oocytes in comparison with infected sperm with NCP BVDV (n ⫽ 125) of fertile group. Nevertheless, the negative effect was also noticeable on sperm attachment capacity (n ⫽ 127) to the ZP of the fertile oocytes infected with CP BVDV biotype, in comparison with infected fertile oocytes with NCP BVDV (n ⫽ 151). There were no significant differences between sperm attachment to the ZP of infertile oocytes of infected male gametes with CP (n ⫽ 104) or NCP (n ⫽ 122) BVDV, as was also true in infertile female gametes infected with CP (n ⫽ 204) or NCP (n ⫽ 195) BVDV. Effects on fertilization rate of the presence of CP
1072
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
Table 1 Rate of successful in vitro fertilization in bovine oocytes incubated with CP and NCP BVDV of sperm combined with oocytes incubated with 106 TCID50/mL of CP or NCP BVDV at two sperm concentrations. Sperm/mL 105 106 a-c
Rate of IVF (%) in control and treatment groups Control
Sperm and CP
Sperm and NCP
Oocytes and CP
Oocytes and NCP
48%a (43/89) 74%a (64/86)
35%a (30/84) 4%c (4/99)
29%b (23/80) 14%c (13/91)
38%a (37/96) 19%c (19/99)
22%b (20/89) 48%c (48/99)
Within a row, percentages without a common superscript differed (P ⬍ 0.05).
and NCP BVDV on sperm and oocytes during the IVF process are shown (Table 1). Compared to the control group, fertilization rates were significantly reduced both in the low (105 sperm/mL) and high (106 sperm/ mL) sperm concentration groups with male and female gametes infected with NCP BVDV biotypes (P ⬍ 0.05). However, fertilization rate did not differ significantly with the low sperm concentration in gametes infected with CP BVDV. However, fertilization rate in infected NCP BVDV sperm (29%) and oocytes (22%) differed (P ⬍ 0.05). Furthermore, fertilization rates were reduced from sperm and oocytes infected with CP BVDV at the higher sperm concentration (P ⬍ 0.05). 4. Discussion In this study, the only treatment that increased sperm binding to the ZP was oocytes infected with CP BVDV at the lower sperm concentration (105sperm/mL). With the higher sperm concentration (106 sperm/mL), the number of sperm binding to the ZP of control treatment oocytes was so high as to be uncountable. In the group of fertilized oocytes, sperm attachment markedly decreased when sperm were infected with CP BVDV in comparison with NCP BVDV. The underlying reasons for the differences in responses of sperm and ova to the CP and NCP BVDV biotypes were unclear. There is little detailed knowledge of the molecular mechanisms responsible for the entry of BVDV biotypes into bovine cells, but several reasons can be postulated for observed differences. Some glycoproteins (gp), especially gp48 and gp53, present in the viral envelope are thought to be involved in attachment of the virus to its host cells [26,27]. Furthermore, although different BVDV biotypes do not necessarily have the same receptor, the presence of multiple receptors for BVDV attachment to the cell surface may contribute to the BVDV attachment to the host cells. It has also been reported that BVDV uses more than one receptor for binding [28]. Some viruses can attach strongly to oocytes [8]; for example, there has recently been better understanding regarding the
mechanism by which Pestiviruses attach to their host cells. Bovine CD46 acts as a cellular receptor for BVDV [29]. Perhaps CP and NCP BVDV use different receptors for cell attachment or entry; further basic investigations are needed to confirm this theory. In the current study, the presence of CP and NCP BVDV during the IVF process with two different sperm concentrations significantly reduced the fertilization rate. The fertilization rate of control group oocytes at the lower sperm concentration was lower than at the higher sperm concentration; this lower fertilization rate could have been due to the 10-fold lower concentration of sperm. Conversely, at the concentration of 105 sperm/mL, fertilization rate of CP BVDV-infected oocytes was not affected. In a previous study, fertilization rate of intact oocytes was reduced only with sperm that had been incubated with CP BVDV [30]. Influences of CP and NCP BVDV in decreasing fertilization rate during IVF were manifested mainly by reduced sperm attachment to the fertile and infertile oocytes at the higher sperm concentration. Therefore, in the control group, sperm attachment to the ZP of oocytes was too high to be counted, due to normal binding of the sperm to the ZP; this further confirmed the negative effects of BVDV on the sperm-oocyte interaction. However, with lower sperm concentration, only fertile oocytes infected with CP BVDV differed in sperm binding to the ZP. Although, it has been shown that the intact ZP of oocytes can act as a penetration barrier against pathogens [31], in oocytes infected with BVDV, this barrier may have been compromised, facilitating sperm penetration during fertilization [32]. The origin of oocytes is of considerable importance in the sanitary risks associated with production of in vitro embryos. Oocytes are surrounded by cumulus cells. The role of the surrounding cumulus cells in protecting IVF embryo from contamination and chemical damage is very important [33,34]. Semen is rarely considered a source of virus transmission [35]. However, viruses can easily bind to the male gamete [20,25,36]. Pathogens such as BVDV can contaminate follicular fluid, granolosa, and cumulus cells, and even
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
cells from the ovarian stroma [37]. Therefore, BVDV biotypes can bind to the ZP and penetrate into the oocytes during fertilization. Most studies reported lower rates of fertilization and embryo development during BVDV infection [38,39], which may depend on BVDV strains, biotypes [21], and the dose of the virus [39]. Inhibition of sperm attachment to the ZP may be due to a toxic or cytopathic influence of BVDV. Perhaps BVDV reduces the ability of sperm to attach to the ZP by decreasing sperm motility (total and progressive), sperm capacitation and the acrosome reaction. The effect of BVDV infection at the time of IVF has been the focus of several experimental studies [40,41]. In France, fertilization and embryo cleavage rates were reduced when semen from a PI bull was used for IVF [40]. In a subsequent study, there were three groups of in vitro-matured oocytes: the first group was exposed to semen from a PI bull (positive control); the second was exposed to the same semen after it had been treated during the ‘swim-up’ phase with a specific anti-BVDV immunoglobulin, and the third group was exposed to semen from a BVDV-free bull (negative control) [41]. Proportions of oocytes developing to the blastocyst in the three groups were 4.0, 8.2, and 13.9%, respectively. The authors suggested that the low rate of embryo development in the first group was a specific effect of the virus. They also claimed that the marginally reduced blastocyst-formation rate in the second group, in which the virus neutralized, may have been due to poor semen quality in the PI bull. The present study confirmed the negative effects of CP and NCP BVDV on sperm attachment on the ZP and fertilization rates. This effect could result mainly from the reduction of sperm attachment to the ZP of oocytes [30]. In a study by Bielanski and Loewen, when a NCP BVDV strain (105 TCID50/mL) was incubated with semen from an uninfected bull prior to IVF, 22% of embryos developed to blastocysts. Moreover, the percentages of oocytes which cleaved and the percentages of embryos which developed to blastocysts were 47, 51, and 20%, and 17, 20, and 13% using semen from the three PI bulls, respectively, and were 53 and 22% using pooled semen from three uninfected bulls [41]. There are considerable data regarding the interaction of BVDV with in vitro-produced embryos, but few data regarding whether semen used for IVF can lead to transfer of BVDV, via sperm, to oocytes during fertilization [30]. The BVDV replicates entirely within the cytoplasm of the host cells. High virus concentrations have been reported in the blood, nasal secretions, sa-
1073
liva, tears, milk, urine, feces, lymph nodes, and reproductive organs of PI cattle. Tissue distribution of BVDV is widespread in clinically infected cattle [42,43,44]. It has also been shown that BVDV infection can be transmitted through the semen of transiently infected bulls under field conditions [45]. The BVDV can be the cause of infertility in the field due to early embryo death, reduction of sperm attachment to the ZP, interference with cells in the reproductive tract (e.g., cumulus and granulosa cells) and follicular fluid performance [46]. Cattle herds can be infected by BVDV. Recently, it has been shown that dairy cattle in Iran were infected with this virus [6,47], which can be an underlying cause of infertility and reproductive problems in dairy herds. In conclusion, both CP BVDV and NCP BVDV interacted with sperm and oocyte surfaces in cattle; this interaction influenced sperm binding to the ZP of oocytes and IVF rate. Oocytes infected with CP or NCP BVDV compromised sperm attachment to the ZP. At the higher level of sperm concentration evaluated in this study, both biotypes of BVDV had greater reduction in sperm attachment to the ZP than at the lower sperm concentration. Regardless, the exact mechanisms of BVDV attachment on male and female gametes need more fundamental investigations. Acknowledgments This study was supported financially by Ferdowsi University of Mashhad, Mashhad-Iran and the Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. The authors gratefully thank Professor Ann Van Soom, Professor Hans Nauwynck, and Professor Aart de Kruif for their support. The virus stock was kindly provided by the Veterinary and Agriculture Research Center, Brussels, Belgium. References [1] Radostits OM, Gay CC. Hinchcliff KW. Constable PD. Veterinary Medicine. 10th Edition, W. B. Saunders, England. 2007, p. 1249 – 60. [2] McClurkin AW, Littledike ET, Cutlip RC, Frank GH, Ceria MF, Bolin SR. Production of cattle immunotolerant to bovine viral diarrhea virus. Can J Comp Med 1984;48:156 – 61. [3] Donis RO. Molecular biology of bovine viral diarrhea virus and its interactions with the host. Vet Clin North Am Food Anim Prac 1995;11:393– 423. [4] Zhang G, Aldridge S, Clarke MC, McCauley JW. Cell death induced by cytopathic bovine viral diarrhoea virus is mediated by apoptosis. J Gen Virol 1996;77:1677– 81.
1074
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075
[5] Lindberg A. The Nordic bovine viral diarrhoea virus eradication programmes—their success and future. Cattle Pract 2004;12: 3–5. [6] Talebkhan Garoussi M, Haghparast A, Estajee H. Prevalence of bovine viral diarrhoea virus antibodies in bulk tank milk of industrial dairy cattle herds in suburb of Mashhad-Iran. Prev Vet Med 2008;84:171– 6. [7] Stringfellow D, Wrathall A. Epidemiological implications of the production and transfer of IVF embryos. Theriogenology 1995;43:89 –96. [8] Bielanski A. A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and related new reproductive technologies. Biotech Adv 1997;15: 633–56. [9] Wrathall AE, Simmons HA, Van Soom A. Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilisation with virus-infected semen. Theriogenology 2006;65:247–74. [10] Iqbal M, McCauley JW. Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis. J Gen Virol 2002;83: 2153–9. [11] Krey T, Himmelrreich A, Heimann M, Menge C, Jurgen H, Maurer K, Rumenapf T. Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1. J Virol 2006;80:3912–22. [12] Liebler TEM, Greiser W, Pohlenz JF. Organ and tissue distribution of the antigen of the cytopathogenic bovine virus diarrhea virus in the early and advanced phase of experimental mucosal disease. Arch Virol 1997;142:1613–34. [13] Cattaneo R. Four viruses, two bacteria and one receptor: membrane cofactor protein (CD46) as pathogens magnet. J Virol 2004;78:4385– 8. [14] Tsuboi T, Imada T. Bovine viral diarrhea virus replication in bovine follicular epithelial cells derived from persistently infected heifers. J Vet Med Sci 1998;60:569 –72. [15] Kirkland PD, Mackintosh SG, Moyle A. The outcome of wide spread use of semen from a bull persistently infected with pestivirus. Vet Rec. 1994;135:527–9. [16] Gard JA, Given MD, Stringfellow DA. Bovine diarrhea virus (BVDV): Epidemiologic concerns relative to semen and embryos. Theriogenology 2007;68:434 – 42. [17] Given MD, Heath AM, Brock KV, Brodersen BW, Carson RL, Stringfellow DA. Detection of bovine viral diarrhea virus in semen obtained after inoculation of seronegative postbubertal bulls. Am J Vet Res 2003;64:428 –34. [18] Talebkhan Garoussi M, Bassami MR, Afshari SE. Detection of bovine viral diarrhea virus using a nested RT-PCR assay in bulk milk samples of dairy cattle herds in suburb of Mashhad-Iran. Iran J Biotechnol 2007;5:52–5. [19] Gard JA, Given MD, Stringfellow DA. Bovine diarrhea virus (BVDV): Epidemiologic concerns relative to semen and embryos. Theriogenology 2007;68:434 – 42. [20] Bielanski A, Dubuc C, Hare WCD. Failure to remove bovine viral diarrhea virus from bull semen by swim-up and other separatory sperm techniques associated with in vitro fertilization. Reprod Dom Anim 1992;27:303– 6. [21] Van Soom A, Vanopdenbosh E, Mahmouzadeh A R, de Kruif A. In vitro production of Belgian Blue cattle embryo: some data on the risk of viral infections. Vlaams Diergenees Kundig Tijdschrift 1994;63:139 – 45.
[22] Mahmoudzadeh AR, Van Soom A, Bols P, Ysebaert M T, de Kruif A. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of development stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival. J Reprod Fertil 1995;103:33–9. [23] Vanroose G, Nauwynck H, Van Soom A, Vanopdenbosch E, de kruif A. Replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona free and zona-intact in vitro produced bovine embryos and the effect on embryo quality. Biol Reprod 1998;58:857– 66. [24] Vanroose G. Interaction of bovine herpesvirus-1 and bovine viral diarrhea virus with bovine gametes and in-vitro produced embryos. Ghent University. Ph. D thesis. 1999;174-5. [25] Tanghe S, Vanroose G, Van Soom A, Duchateau L, Ysebaert MT, Kerkhofs P, Thiry E, Van Drunen Little-van den Hurk S, Van Oostveldt P, Nauwynck H. Inhibition of bovine–zona binding by bovine herpesvirus-1. Reproduction 2005;130:251–9. [26] Donis RO, Dubovi EJ. Glycoproteins of bovine viral diarrhea– mucosal disease virus in infected bovine cells. J Gen Virol 1987;68:1607–16. [27] Boulanger D, Waxweiler S, Karelle L, Loncar M, Mignon B, Dubuisson Thiry E, Pastoret PP. Characterization of monoclonal antibodies to bovine viral diarrhea virus: evidence of a neutralization activity against gp48 in the presence of antimouse immunoglobulin serum. J Gen Virol 1991;72:1389 – 402. [28] Schelp C, Greiser-Wilke I, Wolf G, Beer M, Moennig V, Liess B. Identification of cell member proteins linked to susceptibility to bovine viral diarrhea virus infection. Arch Virol 1995;140: 1997–2009. [29] Maurer KT, Krey V, Moennig H, Thiel J, Rumenapf T. CD46 is a cellular receptor for bovine viral diarrhea virus. J Virol 2004; 78:1792–9. [30] Talebkhan Garoussi M. The effects of cytopathic and noncytopathic Bovine Viral Diarrhoea Virus with sperm cells on in vitro fertilization of bovine oocytes. Vet Res Commun 2007;31:365– 70. [31] Vanroose G, Nauwynck H, Van Soom A, Ysebaert MT, Charlier G, Van Oostveldt P. Structural aspects of the zona pellucida of in-vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biol Reprod 2000; 62:463–9. [32] Le Tallec B, Ponsart C, Marquant-Le Guienne B, Guérin B. Risks of transmissible diseases in relation to embryo transfer. Reprod Nutr Develop 2001;41:439 –50. [33] Stringfellow DA, Seidel S. Manual of the International Embryo Transfer Society. IETS, Urbana, Illinois. 1998. [34] Tanghe S, Van Soom A, Mehrzad J, Maes D, Duchateau L, de Kruif A. Cumulus contributions during bovine fertilization in vitro. Theriogenology 2003;60:135– 49. [35] Bielanski A. The problems and risks of the contamination of germplasm with pathogenic microbes using current reproductive technologies. In: Enne V, Greppi E, Lauria A, editors. Reproduction and Animal Breeding: Advances and Strategy. Elsevier, 1995, p. 99 –116. [36] Tsuboi T, Osawa T, Kimura K, Kubo M, Haritani M. Experimental infection of early pregnant cows with bovine viral diarrhea virus: Transmission of virus to the reproductive tract and conceptus. Res Vet Sci 2010 (In press). [37] Booth P J, Stevens DA, Collins ME, Brownlie J. Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle. J Reprod Fertil 1995;105:17–24.
M. Talebkhan Garoussi and J. Mehrzad / Theriogenology 75 (2011) 1067–1075 [38] Guérin B, Chaffaux S, Marquant-Leguienne B, Allietta M, Thibier M. IVF and IV culture of bovine embryos using semen from a bull persistently infected with BVD. Theriogenology 1992;37:217. [39] Vanroose G, Nauwynck H, Van Soom A, Vanopdenbosch E, de Kruif A. Effect of bovine herpesvirus-1 or bovine viral diarrhea virus on development of in vitro-produced bovine embryos. Mol Reprod Dev 1999;54:255– 63. [40] Allietta M, Guerin B, Marquant-Le GB, Thibier M. The effect of neutralization of BVD/MD virus present in bovine semen on the IVF and development of bovine embryos. Theriogenology 1995;43:156. [41] Bielanski A, Loewen K. In vitro fertilization of bovine oocytes with semen from bulls persistently infected with bovine viral diarrhoea virus. Anim Reprod Sci 1994;35:183–9. [42] Avery B, Greve T, Ronsholt L, Botner A. Virus screening of a bovine in vitro embryo production system. Vet Rec 1993;132: 660.
1075
[43] Baszler TV, Evermann JF, Kaylor PS, Byington TC, Dilbeck PM. Diagnosis of naturally occurring bovine viral diarrhea virus infections in ruminants using monoclonal antibody-based immunohistochemistry. Vet Pathol 1995;32:609 –18. [44] Brownlie J. The pathways for bovine virus diarrhoea virus biotypes in the pathogenesis of disease. Arch Virol 1991;3:79 – 96. [45] Rikula U, Nuotio L, Laamanen UI, Sihvonen L. Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions. Vet Rec 2008; 162:79 – 81. [46] Daniel LG. Reproductive consequences of infection with Bovine Viral Diarrhea Virus. Vet Clin Food Anim 2004;20: 5–19. [47] Talebkhan Garoussi M, Haghparast A, Hajenejad MR. Prevalence of Bovine Viral Diarrhoea Virus antibodies among the industrial dairy cattle herds in suburb of Mashhad-Iran. Trop Anim Health Prod 2009;41:663–7.