The effect of bovine pestivirus infection on the superovulatory response of Friesian heifers.

The pathogenesis of reproductive loss associated with bovine pestivirus infection during the preovulatory period was investigated using superovulated heifers. Twenty-five Friesian heifers were selected and randomly assigned to either a control group (n = 12) which did not become infected or to a treatment group (n = 13) which became infected following intranasal instillation of 2 ml of serum inoculum containing 5.5 log(10) TCID(50)/ml non-cytopathic virus, 9 d prior to artificial insemination (AI). Transrectal ultrasonography was used to monitor follicular development and ovulation during the superovulatory period. Animals were superovulated using a standard protocol of twice-daily injections of FSH-P and then were inseminated twice commencing 12 h after the onset of estrus. The intensity of expression of estrus was higher in the control heifers than in the pestivirus-infected heifers. Of 13 pestivirus-infected heifers, only 3 heifers displayed standing estrus compared with that in the control group, in which 10 of 12 heifers exhibited standing estrus. The mean number of ova/embryos recovered from the control group heifers was 5.75 +/-2.31, of which 4.00 +/- 0.72 were evaluated as transferable quality embryos. In comparison, heifers in the pestivirus-infected group yielded only a mean of 0.60 +/-0.34 ova/embryos, of which 0.23 +/- 0.22 were transferable quality embryos. Based on ultrasonographic examination, 24 h after the first AI 82% of the presumptive ovulatory follicles had ovulated in the control group compared with an ovulation rate of only 17% in the treated group. The results of this experiment demonstrated that bovine pestivirus infection during the preovulatory period could adversely affect ovulation, thus leading to a significant reduction in the number of palpable corpora lutea and in the number and quality of embryos recovered.

Factors that influence follicle recruitment, growth and ovulation during ovarian superstimulation in heifers: opportunities to increase ovulation rate and embryo recovery by delaying the exposure of follicles to LH.

The outcome of ovarian follicular superstimulation protocols in heifers is influenced by the number of follicles that are stimulated to grow and the number induced to ovulate. At present, only a proportion of the follicles that are stimulated to grow progress to ovulation. The argument is developed in this review that failure of some of these follicles to ovulate may be due not to an intrinsic deficiency but rather to their relatively small size when the FSH treatment is initiated. Consequently, these follicles do not have the opportunity to undergo full maturation within the time frame of a conventional superstimulation protocol Based on this argument, we propose that delaying the LH surge would allow for completion of maturation by a greater number of follicles, resulting in an increased ovulation rate and in recovery of a greater number of viable embryos.

Use of a GnRH agonist to prevent the endogenous LH surge and injection of exogenous LH to induce ovulation in heifers superstimulated with FSH: a new model for superovulation.

A new protocol for superovulating cattle which allows for control of the timing of ovulation after superstimulation with FSH was developed. The preovulatory LH surge was blocked with the GnRH agonist deslorelin, and ovulation was induced by injection of LH. In Experiment 1, heifers (3-yr-old) were assigned to a control group (Group 1A, n = 4) or a group with deslorelin implants (Group 1B, n = 5). On Day -7, heifers in Group 1A received a progestagen CIDR-B((R))device, while heifers in Group 1B received a CIDR-B((R))device + deslorelin implants. Both groups were superstimulated with twice daily injections of FSH (Folltropin((R))-V): Day 0, 40 mg (80 mg total dose on Day 0); Day 1, 30 mg; Day 2, 20 mg; Day 3, 10 mg. On Day 2, heifers were given PGF (a.m.) and CIDR-B((R)) devices were removed (p.m.). Three heifers in Group 1A had a LH surge and ovulated, whereas neither of these events occurred in Group 1B (with deslorelin implants) heifers. In Experiment 2, heifers (3-yr-old) were assigned to 1 of 4 equal groups (n = 6). On Day -7, heifers in Group 2A received a norgestomet implant, while heifers in Groups 2B, 2C and 2D received norgestomet + deslorelin implants. Heifers were superstimulated with FSH starting on Day 0 as in Experiment 1. On Day 2, heifers were given PGF (a.m.) and norgestomet implants were removed (p.m.). Heifers in Groups 2B to 2D were given 25 mg LH (Lutropin((R))): Group 2B, Day 4 (a.m.); Group 2C, Day 4 (p.m.); Group 2D, Day 5 (a.m.). Heifers in Group 2A were inseminated at estrus and 12 and 24 h later, while heifers in Groups 2B to 2D were inseminated at the time of respective LH injection and 12 and 24 h later. Injection of LH induced ovulation in heifers in Groups 2B to 2D. Heifers in Group 2C had similar total ova and embryos (15.2 +/- 1.4) as heifers in Group 2A (11.0 +/- 2.8) but greater (P < 0.05) numbers than heifers in Group 2B (7.0 +/- 2.3) and Group 2D (6.3 +/- 2.0). The number of transferable embryos was similar for heifers in Group 2A (5.8 +/- 1.8) and Group 2C (7.3 +/- 2.1) but lower (P < 0.05) for heifers in Group 2B (1.2 +/- 0.8) and Group 2D (1.3 +/- 1.0). The new GnRH agonist-LH protocol does not require observation of estrus, and induces ovulation in superstimulated heifers that would not have an endogenous LH surge.

Close synchrony of ovulation in superstimulated heifers that have a downregulated anterior pituitary gland and are induced to ovulate with exogenous LH.

The synchrony of ovulation was examined in superstimulated heifers that had a downregulated pituitary gland and which were induced to ovulate by injection of exogenous LH. The pituitary was downregulated and desensitized to GnRH by treatment with the GnRH agonist deslorelin. Nulliparous heifers (3.5 yr old) at random stages of the estrous cycle were assigned to 1 of 3 groups, and on Day -7 received the following treatments: Group 1 (control, n = 8), 1 norgestomet ear implant; Group 2 (GnRH agonist, n = 8); Group 3 (GnRH agonist-LH protocol, n = 8), 2 deslorelin ear implants. Ovarian follicle growth in all heifers was superstimulated with twice-daily intramuscular injections of FSH (Folltropin-V): Day O, 40 mg (80 mg total dose); Day 1, 30 mg; Day 2; 20 mg; Day 3, 10 mg. On Day 2, all heifers were given a luteolytic dose of PGF (7 A.M.), Norgestomet implants were removed from heifers in Group 1 (6 P.M.). Heifers in Group 3 were given an injection of 25 mg, i.m. porcine LH (Lutropin) on Day 4 (4 P.M.). Ovarian follicle status was monitored at 8-h intervals from Day 3 (8 A.M.) to Day 6 (4 P.M.) using an Aloka Echo Camera and 7.5 MHz transducer. Heifers in Groups 2 and 3 exhibited estrus earlier (P < 0.05) than heifers in Group 1. Heifers in Group 2 did not have a preovulatory LH surge and they did not ovulate. Individual control heifers in Group 1 ovulated between 12 A.M. on Day 5 and 8 A.M. on Day 6. Heifers with deslorelin implants and injected with LH in Group 3 ovulated between 4 P.M. on Day 5 and 8 A.M. on Day 6. It was confirmed that superstimulated heifers with GnRH agonist implants can be induced to ovulate with LH. It was also demonstrated that ovulation is closely synchronized after injection of LH. Thus, a single, fixed-time insemination schedule could be used in a GnRH agonist-LH superovulation protocol, with significant practical and economic advantages for superovulation and embryo transfer programs.

Studies of the pathogenesis of bovine pestivirus-induced ovarian dysfunction in superovulated dairy cattle.

Two experiments (Experiment I, n=12 Holstein-Friesian heifers; Experiment II, n=8 Jersey cows) were conducted to investigate the pathogenesis of bovine pestivirus-induced ovarian dysfunction in cattle. In both experiments the cattle were superovulated with twice daily injections of a porcine pituitary extract preparation of follicle stimulating hormone (FSH-P), for 4 days commencing on Day 10+/-2 after a presynchronised oestrus. The heifers received a total dose of 30 mg and the cows 32 mg of FSH-P. Prostaglandin F(2alpha) (PGF(2alpha)) was administered 48 h after commencement of superovulation and all cattle were artificially inseminated (AI) between 48 and 66h after PGF(2alpha) treatment. In both experiments bovine pestivirus seronegative cattle (Experiment I, n=6; Experiment II, n=4) were inoculated intranasally with an Australian strain of non-cytopathogenic bovine pestivirus (bovine viral diarrhoea virus Type 1) 9 days prior to AI. Bovine pestivirus infection was confirmed by seroconversion and/or virus isolation in all of the inoculated cattle, consistent with a viremia occurring approximately between Day 5 prior to AI and the day of AI. Ovarian function was monitored in both experiments by daily transrectal ultrasonography and strategic blood sampling to determine progesterone, oestradiol-17beta, luteinising hormone (LH) and cortisol profiles. Non-surgical ova/embryo recovery was performed on Day 7 after AI. In Experiment II half the cattle were slaughtered on Day 2 and the remainder on Day 8 after AI, and the ovaries submitted for gross and histopathological examination including immunohistochemistry to demonstrate the presence of bovine pestivirus antigen. In both studies, comparisons were made between infected and confirmed uninfected (control) animals. Overall the bovine pestivirus infected cattle had significantly lower (P<0.05) ova/embryo recovery rates compared to the control cattle. There was evidence of either an absence (partial or complete) of a preovulatory LH surge or delay in timing of the LH peak in the majority (90%) of infected heifers and cows, and histologically, there was evidence of non-suppurative oophoritis with necrosis of granulosa cells and the oocyte in follicles from the infected cows. By contrast only 20% of the control heifers and cows had evidence of absence of a pre-ovulatory LH surge. These experiments collectively demonstrate that bovine pestivirus infection during the period of final growth of preovulatory follicles may result in varying degrees of necrosis of the granulosa cells with subsequent negative effects on oestradiol-17beta secretion which in turn negatively affects the magnitude and/or timing of the preovulatory LH surge.

Transcervical transfer of bovine embryos.

Six-to nine-day-old embryos collected either surgically or nonsurgically from superovulated donor cows were transferred transcervically to recipient cows. Polyethylene tubes and plastic insemination pipettes were used to transfer the embryos. When embryos were transferred under controlled conditions (laboratory transfer) to 12 recipients, 10 (83.3 per cent) and eight (66.7 per cent) were tested pregnant at 45 and 90 days after transfer respectively. When transfers were made to 12 recipients under field conditions (paddock transfer), nine (75 per cent) and five (41.7 per cent) were tested pregnant at 45 and 90 days after transfer respectively. The difference in pregnancy rates between laboratory and paddock transfers was not statistically significant.

Techniques of surgical and non-surgical ova collection of superovulated cows.

A comparison is made of the results of ova collection from 65 superovulated cows of varying ages using the conventional surgical technique and a non-surgical (transcervical) technique. Two types of apparatus, rigid and flexible, were developed for non-surgical collections. The problems associated with these techniques are discussed and some remedial measures suggested. A mean of 9.8 ova per donor cow were recovered by the surgical method compared to 2.9 and 3.0 ova recovered by the transcervical method using flexible and rigid apparatus respectively. Eight young cows, which had not been previosly subjected to superovulation and embryo collection, yielded an average of 5.6 ova per donor by the non-surgical technique.

Unsuccessful attempts to collect ova by surgical fixation of uterine catheters in superovulated cows.

Attempts were made to collect ova from superovulated cows by surgically fixing indwelling silastic balloon catheters in the uterine lumen. No ova were collected from the four catheterised cows and it was shown that ovarian activity was depressed. In this group, only 16 ovulations occurred compared with six control cows in which a total of 121 ovulations were recorded and 84 ova were collected. Also the ovaries of the catheterised cows had six large cysts, whereas no cysts were recorded in the control cows. The catheters flushed perfectly and bacteriological cultures of the flushing and uteri showed that no infections had occurred. The cows tolerated the catheters extremely well. There was no depression in appetite nor was any abnormal behaviour recorded. However, the severe depression of ovulation and the formation of ovarian cystic follicles prevents the technique from having any practical application as a means of collecting ova.

Successful transfer of a bovine embryo through a cannulated fallopian tube.

The fallopian tubes of a recipient cow were surgically cannulated and the cannulae brought out through the flank. Two fertilised eggs, recovered surgically from a donor cow, were transferred to the uterus of the recipient cow through the cannulae. The recipient became pregnant and gave birth to a live normal female calf 289 days after the transfer. After the birth of the calf one cannula was found to be patent.

Preparation of teaser bulls by dorsal scrotal penile deflection.

A simple, quick and reliable technique of preparing teaser bulls has been developed. Four Bos indicus aged between 1 year 6 months and 2 years were subjected to this method by deflecting their penes backwards about 2 to 3 cm posterior and dorsal to the attachment of the scrotum. No serious postoperative complications were recorded. The sexual behaviour and libido of the bulls did not change after subjecting them to this technique.