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Effect of time of ovulation on fertilization after intrabursal transfer of spermatozoa (ITS): improvement of a new method for artificial insemination in mice.

The timing of AI in relation to ovulation was examined to improve intrabursal transfer of spermatozoa (ITS) in mice, a new method of AI that involves transfer of spermatozoa into a space near the infundibulum. Two microliters of fresh epididymal B6C3F1 spermatozoa (containing 2 x 10(5) spermatozoa) were inseminated 1, 7, 12, or 17 h after hCG administration. At 1.7 days after ITS, normal cleaving embryos were recovered at rates ranging from 6 to 50% (21.5 +/- 15.8%; mean +/- S.D.), 40-100% (75.2 +/- 20.2%), 33-100% (60.1 +/- 19.3%), and 6-47% (22.7 +/- 13.3%), respectively. The rate obtained by ITS 7h after hCG administration was comparable (P > 0.05) to that (90.5 +/- 6.3%) for embryos obtained after natural mating (control), but rates at all other times were significantly less than control. To examine whether in vivo fertilization rate differs when spermatozoa from various mouse strains are used, B6C3F1 females were inseminated with spermatozoa from ICR, C57BL/6N and C3H/HeN mice 7 h after hCG administration. There were strain differences (P < 0.01 for ICR and B6C3F1 versus C57BL/6N and C3H/HeN) for in vivo fertilization rates (83.9 +/- 10.3%, 75.2 +/- 20.2%, 33.6 +/- 24.5% and 25.6 +/- 16.1% for ICR, B6C3F1, C57BL/6N and C3H/HeN, respectively). Similar rates (72.9 +/- 7.3% and 27.5 +/- 46.2% for ICR and C57BL/6N, respectively) were also obtained when oocytes were inseminated with spermatozoa of the same strain. In addition, females (B6C3F1) inseminated by ITS of fresh B6C3F1 spermatozoa 7 h after hCG administration yielded normal mid-gestational fetuses with an average litter size of 7.0 +/- 4.9, which seemed much higher than the previously reported litter size of 3.2. In conclusion, the timing of AI was considered a key factor affecting in vivo fertilization efficiency.

Animals↗

Effect of presynchronization using prostaglandin F2alpha and a milk-ejection test on pregnancy rate after the timed artificial insemination protocol, Ovsynch.

The objective of this research was to determine if PGF2alpha-induced milk letdown (ML) is an accurate indicator of luteolysis, allowing cows to be synchronized to begin the Ovsynch protocol (GnRH-7d-PGF2alpha-2d-GnRH-24h-AI) at the most beneficial time of the estrous cycle (days 5-9), and determine if this would improve pregnancy rate (PR). Lactating Holstein cows between 55 and 70 days in milk were used to evaluate the ML test and PR after the Ovsynch protocol, when initiated on the basis of the test result (PROSYNCH). PROSYNCH cows (n = 60) had one teat cannulated to test for ML and were treated with 500 microg cloprostenol, PGF2alpha analogue (PG). Cows with ML were started on Ovsynch 10 days later, and those without started 3 days later. Cows in the control group (OVSYNCH, n = 64) were injected with physiological saline and observed for ML. This group was started on Ovsynch 10 days after saline treatment. Milk samples were collected thrice weekly to determine progesterone concentrations. ML indicated luteolysis with a sensitivity of 98% and a specificity of 60%. The positive and negative predictive values were 83 and 92%, respectively. Pregnancy rates were 48% for PROSYNCH and 52% for OVSYNCH (P = 0.72). When data from both groups were combined, PR was greater in cows that started the Ovsynch protocol in stage 2 of the estrous cycle (days 5-9, 67%) than all other stages (stage 1: days 1-4, 35%; stage 3: days 10-16, 45%; stage 4: days 17-21, 42%; P < 0.01). The proportion of animals with ovulation after GnRH#1, luteolysis after PGF2alpha, and ovulation after GnRH#2 were all greater in the PROSYNCH group (77% versus 55%, P < 0.02; 83% versus 66%, P < 0.03; 97% versus 84%, P < 0.03, respectively). Therefore, the ML test indicated luteolysis with sufficient precision to time the initiation of the Ovsynch protocol between days 5 and 9 of the cycle, however, this did not alter PR compared to starting the protocol randomly throughout the cycle. Initiating the Ovsynch protocol between days 5 and 9 of the cycle increased PR, and improved the efficacy of each injection.

Animals↗

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination.

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.

Animals↗

Viruses in boar semen: detection and clinical as well as epidemiological consequences regarding disease transmission by artificial insemination.

Many viruses have been reported to be present in boar semen, particularly during the viremic phase of the diseases. Some of them, such foot-and-mouth disease virus, porcine reproductive and respiratory syndrome virus, swine vesicular disease virus, porcine parvovirus, picornaviruses, adenoviruses, enteroviruses, Japanese encephalitis virus, pseudorabies virus, African swine fever virus and reoviruses are of particular importance and accurate monitoring prior to and during the presence of boars in AI stations is essential. Various methods may be used to detect these viruses in the animals, or even directly in batches of semen. Cell culture, ELISA and PCR are the most accurate and widely used. Because of the high risk of dissemination of disease via AI, the absolute goal is to provide pathogen-free semen and this is feasible with the adequate measures that are discussed briefly in this paper.

Animals↗

Efficacy of the Ovsynch protocol for synchronization of ovulation and fixed-time artificial insemination in Murrah buffaloes (Bubalus bubalis).

Two experiments were conducted to assess the timing and synchrony of ovulation, plasma LH concentrations, and pregnancy rate in Murrah buffaloes (Bubalus bubalis) treated with the Ovsynch (GnRH-PGF(2 alpha)-GnRH) protocol. In Experiment 1, 10 non-lactating cycling buffaloes received 10 microg of a GnRH analogue i.m. (buserelin acetate) without regard to the stage of the estrous cycle (day of treatment, day 0), followed by 25mg of PGF(2 alpha) i.m. (dinoprost thromethamine) 7 days later. A second-treatment of the same GnRH analogue (10 microg, i.m.) was given 48 h after PGF(2 alpha). Ovulation was confirmed by transrectal palpation (at 2-h intervals) from the second-GnRH treatment to detection of ovulation or up to 96 h after the second-GnRH treatment. Plasma LH concentrations were determined in blood samples collected at 15-min intervals for 6h, starting at the second-GnRH treatment, and thereafter at 2-h intervals until 2h after detection of ovulation. Ovulation occurred in 9/10 buffalo (90%) 23.3+/-1.3h (mean+/-S.E.M.; range 20--32 h) after the second-GnRH treatment. Peak LH concentrations 13.5+/-3.5 ng/mL (range 3.9--40.0 ng/mL) occurred 2.1+/-0.1h (range 1.2-3.0 h) after the second-GnRH treatment. In Experiment 2, 15 lactating, cycling buffaloes were subjected to the Ovsynch protocol, with fixed-time AI 12 and 24h after the second-GnRH treatment and 75 lactating buffaloes were inseminated, approximately 12h after detection of spontaneous estrus. Pregnancy rates were 33.3% for TAI and were 30.7% for buffaloes inseminated following spontaneous estrus (P=0.84). In conclusion, the Ovsynch protocol effectively synchronized ovulation in Murrah buffaloes and resulted in conception rates (to two fixed-time inseminations) that were comparable to those achieved with a single AI after detection of spontaneous estrus.

Animals↗

Comparison of reproductive performance by artificial insemination versus natural service sires in California dairies.

This study compared the calving to conception intervals for cows in AI pens with cows exposed to natural service sires, controlling for milk production, mastitis occurrence, parity and calving month effects. Records from 10 western United States dairy herds (mean herd size = 2058 cows) were evaluated retrospectively over an 18-month period. Eight bull breeding analysis cohorts were created (the first cohort 0-50 days in milk and the remaining cohorts at 25 days in milk intervals through 226 days). The cohorts contained non-pregnant cows that were first moved into bullpens during the described cohort period. Equal numbers of non-pregnant cows only exposed to AI during the cohort period were randomly selected from the pool of eligible non-pregnant cows. An AI cow was used only once in the data analysis, but was included in a bull breeding cohort at a later date if she remained non-pregnant and was transferred to a bullpen. Univariate and multivariate survival analysis was used to compare the calving to conception intervals. Cows in AI groups had higher hazard rates for pregnancy across all cohorts. Parity and milk production were significantly associated with risk for pregnancy. In herds that practice a mixture of AI and bull breeding, overall herd reproductive performance might be improved by allowing cows more opportunities at AI prior to moving them into clean-up bullpens.

Animals↗

Comparison of artificial insemination versus embryo transfer in lactating dairy cows.

Conception rates (CR) are low in dairy cows and previous research suggests that this could be due to impaired early embryonic development. Therefore, we hypothesized that CR could be improved by embryo transfer (ET) compared with AI. During 365 days, 550 potential breedings were used from 243 lactating Holstein cows (average milk production, 35 kg/day). Cows had their ovulation synchronized (GnRH-7d-PGF(2alpha)-3d-GnRH) and they were randomly assigned for AI immediately after the second GnRH injection (Day 0) or for transfer of one embryo 7 days later. Circulating progesterone concentrations and follicular and luteal size were determined on Days 0 and 7. Pregnancy diagnosis was performed on Days 25 or 32 and pregnant cows were reevaluated on Days 60-66. Single-ovulating cows with synchronized ovarian status had similar CR on Days 25-32 with ET (n = 176; 40.3%) and AI (n = 160; 35.6%). Pregnancy loss between Days 25-32 and 60-66 also did not differ (P = 0.38) between ET (26.2%) and AI (18.6%). When single (n = 334) and multiple (n = 57) ovulators were compared, independent of treatment, multiple ovulators had greater (P < 0.001) circulating progesterone concentrations on Day 7 (2.7 ng/ml versus 1.9 ng/ml) and there was a tendency (P = 0.10) for a greater CR in multiple ovulators (50.9% versus 38.1%). However, there was no difference in CR between AI and ET cows with multiple ovulations (50.0% versus 51.7%). In single-ovulating cows, CR tended to be lower for AI than ET in cows ovulating smaller follicles (diameter < or = 15 mm; 23.7% versus 42.3%; P = 0.06) but not average-diameter follicles (16-19 mm; 41.2% versus 37.3%; P = 0.81) or larger (> or =20 mm; 34.3 versus 51.0%; P = 0.36) follicles. Thus, although ET did not improve overall CR in lactating cows, follicle diameter and number of ovulating follicles may determine success with these procedures.

Animals↗

Prevalence of chlamydiae in boars and semen used for artificial insemination.

Although there are indications for venereal transmission of chlamydiae in pigs, direct diagnostic evidence on the presence of these bacteria in boars and boar semen in particular is still incomplete. We investigated boars from two studs (A, B) in semen (A: n = 174; B: n = 100) and faeces (A: n = 174; B: n = 24) for chlamydiae using ompA-PCR and partial ompA gene sequencing. Additionally, blood serum was examined for chlamydial antibodies using an indirect ELISA (A: n = 171; B: n = 62). Chlamydiae were found in 9 (5.2%) and 24 (24.0%) semen specimens, and in 71 (40.1%) and 2 (8.3%) faecal samples from boars of stud A and B, respectively. Regarding individual chlamydial species, Chlamydophila psittaci and Chlamydia suis were identified most frequently, with the former predominating in semen (in 23 out of 33 positive samples) and the latter in faeces (68/73). In contrast, Chlamydophila pecorum was found only sporadically. Chlamydial antibodies were detected in 80 (46.8%) and 6 (9.7%) boars of stud A and B, respectively. No correlation was observed between the data from serology and PCR of semen or faeces in either of the studs. In conclusion, detection of chlamydiae in semen of boars suggests a potential for venereal transmission. Whether the high overall prevalence of chlamydial infections reflects a general situation in boars needs to be investigated. Serological testing failed to identify boars shedding chlamydiae in their semen.

Animals↗

Current status of artificial insemination with donor semen.

As male infertility secondary to oligospermia and azoospermia reamins refractory to treatment, donor insemination has gained greater acceptance. Wives of azoospermic and severely oligospermic men were evaluated for donor insemination. One hundred and thirty-four patients were found to be ovulating normally and to be free from pelvic pathology.Sixty-five accepted donor insemination. Fifty-one conceptions accurred in 41 patients, resulting in 43 live births and 8 miscarriages. The mean number of inseminations was 4.2 (range 1 to 13). Eighty-three patients were found to have endocrinopathies or pelvic pathology interfering with fertility; 58 were considered candidates for corrective therapy to be followed by donor insemination. Conception rates approached those observed in the average population; however, there was a greater incidence of abortion and ectopic gestation. Careful consideration of the patient prior to donor inseminationensures greater success and avoids unnecessary procedures when the patient is not considered a candidate for corrective therapy.

Abortion, Spontaneous↗