New estrus synchronization and artificial insemination protocol for goats based on male exposure, progesterone and cloprostenol during the non-breeding season

This study assesses the effectiveness of a method designed to induce and synchronize ovulation in goats during the non-breeding season, allowing for systematic timed artificial insemination (AI), without the need for prior estrus detection. This method (IMA.PRO2) induces ovulation through the "male effect" and a single 25 mg dose of progesterone given at the time of buck exposure, and early lysis of the induced corpus luteum by the administration of 75 microg of cloprostenol 9 days later. The method was tested in three separate experiments. In experiment 1, estrus was detected in 87.5% of the treated goats 37.0 +/- 1.4 h after cloprostenol administration, with the preovulatory LH surge occurring 40.5 +/- 1.6 h after the cloprostenol injection. In experiment 2, data from 503 does revealed no significant differences in fertility rates between two groups inseminated 48 h (65.5+/-4.0%) or 52 h (63+/-3.0%) after receiving cloprostenol. In experiment 3, 2184 does, comprising 37 replicate groups on 12 farms, were randomly assigned to two trial subgroups. Does in the first subgroup were treated with the IMA.PRO2 method and goats from the second group were given intravaginal progestagens for 11 days, plus 350 IU of eCG and 75 microg of cloprostenol on Day 9 of this treatment. Goats from both subgroups were cervically inseminated at the same time, 50 h after cloprostenol administration in the first group and 46 h after sponge removal in the second. The pregnancy rate achieved with the new method was 64.6%, significantly higher than the yield observed for the use of progestagens plus eCG (46.8%, P<0.01). The simple method proposed as an alternative to the use of progestagen-eCG treatment provides good pregnancy rates to AI undertaken at a fixed time point, and reduces the amount of hormone needed to synchronize estrus in the animals.     

Highly synchronous and fertile reproductive activity induced by the male effect during deep anoestrus in lactating goats subjected to treatment with artificially long days followed by a natural photoperiod

The response to the male effect was studied in two flocks of Saanen and three of Alpine goats during deep anoestrus in three consecutive years. Males and females were subjected to artificially long days for about 3 months (between December 4 and April 1) followed by a natural photoperiod. Bucks joined goats 42-63 days after the end of the long days treatment (between April 20 and June 3) and fertilisation was ensured by natural mating. In experiment 1 (n=248), female goats were treated or untreated with melatonin at the end of the long days treatment and treated or untreated for 11 days with fluorogestone acetate (FGA) before teasing. The males received melatonin implants. In experiment 2 (n=337), the factor studied was the association or non-association of the 11-day FGA treatment. Neither males nor females received melatonin implants. In experiment 3 (n=180), goats were treated for 11 days with FGA or with natural progesterone (CIDR). Neither males nor females received melatonin implants. In experiment 1, among the non-cycling goats (n=218), 99% ovulated and 81% kidded at 161+/-8 days after joining. Ninety-two percent of FGA-treated goats displayed an LH surge at 65+/-11h after teasing. Melatonin treatment did not affect any parameter but FGA advanced the kidding date. In experiment 2, 94% of the goats ovulated and 87% kidded. A major peak of conception was observed on days 3 and 8 after joining in FGA-treated and untreated goats, respectively. Among the FGA-treated goats, 83% displayed an LH surge. Over all flocks, most of the LH surges occurred over a 24-36 h interval, but the surge was initiated at different times in different flocks (36, 48 or 60 h after joining). FGA treatment did not influence the results, except for advancement of births of about 5 days. Differences among flocks were highly significant. In experiment 3, 94% of the goats displayed the LH surge, 93% ovulated and 68% kidded. Significant differences were found among flocks, but not between the FGA and CIDR groups. Bucks marked 85% of the goats 24-72 h after joining. The time interval between the detection of marked goats and detection of the LH surge depended on the time of marking (r=-0.62; p<0.05). In conclusion, treatment of both males and females goats with artificially long days followed by a natural photoperiod is very effective in inducing highly synchronous and fertile reproductive activity via the male effect in the middle of seasonal anoestrus.     

Distribution and number of spermatozoa in the oviduct of the golden hamster after natural mating and artificial insemination

A group of female hamsters was mated with males of proven fertility either several hours before or during ovulation. Another group of females was artificially inseminated several hours before ovulation. Females were killed at various times after the onset of mating or artificial insemination, oviducts were fixed and sectioned serially, and spermatozoa were counted individually as to their location in the oviduct. Regardless of the type or time of insemination, the vast majority of spermatozoa that entered the oviduct remained in the lower segments of the isthmus (the intramural and caudal isthmus) without ascending to the ampulla. The lower segments of the oviduct, particularly the caudal isthmus, appeared to be acting as a "sieve" and/or "sperm reservoir." In females mated or artificially inseminated prior to ovulation, virtually no spermatozoa reached the cephalic isthmus or ampulla until the commencement of ovulation. Although a few spermatozoa reached the ampulla by 1 h after the onset of mating, they were the exception rather than the rule. When females were mated during ovulation, spermatozoa spent a minimum of about 3 h in the caudal isthmus before ascending to the ampulla. The number of spermatozoa that entered the oviduct after artificial insemination was considerably lower than in naturally mated animals, but this low number was apparently large enough to ensure complete fertilization.     

Comparison of controlled internal drug release device and melengesterol acetate as progestin sources in an estrous synchronization protocol for beef heifers

The objectives of this experiment were to compare estrous synchronization responses and AI pregnancy rates of beef heifers using protocols that included either CIDR or MGA as the progestin source. The hypotheses tested were that: (1) estrous synchronization responses after (a) progestin removal, and (b) PGF(2alpha); and, (2) AI pregnancy rates, do not differ between heifers synchronized with either progestin source. At the start of the experiment (Day 0) in both years, heifers were assigned randomly to receive, MGA supplement for 14 days (MGA-treated; n=79) or CIDR for 14 days (CIDR-treated; n=77). On Day 14 progestin was removed and heifers were observed for estrus up to and after PGF(2alpha) on Days 31 and 33 for CIDR-treated and MGA-treated heifers, respectively. Heifers that exhibited estrus within 60h after PGF(2alpha) were inseminated by AI 12h later; the remaining heifers were inseminated at 72h after PGF(2alpha) and given GnRH (100mug). More (P<0.05) CIDR-treated heifers exhibited estrus within 120h after progestin removal than MGA-treated heifers. Intervals to estrus after progestin removal were shorter (P<0.05) for CIDR-treated heifers than MGA-treated heifers. More (P<0.05) CIDR-treated heifers exhibited estrus and were inseminated within 60h after PGF(2alpha) than MGA-treated heifers. Pregnancy rates did not differ (P>0.10) between MGA-treated (66%) and CIDR-treated (62%) heifers. In conclusion, the use of CIDR as a progestin source in a 14-day progestin, PGF(2alpha), and timed AI and GnRH estrous synchronization protocol was as effective as the use of MGA to synchronize estrus and generate AI pregnancies in beef heifers.     

The effects of different insemination regimes on fertility in mares

This study investigated the effects of different artificial insemination (AI) regimes on the pregnancy rate in mares inseminated with either cooled or frozen-thawed semen. In essence, the influence of three different factors on fertility was examined; namely the number of inseminations per oestrus, the time interval between inseminations within an oestrus, and the proximity of insemination to ovulation. In the first experiment, 401 warmblood mares were inseminated one to three times in an oestrus with either cooled (500 x 10(6) progressively motile spermatozoa, stored at +5 degrees C for 2-4 h) or frozen-thawed (800 x 10(6) spermatozoa, of which > or =35% were progressively motile post-thaw) semen from fertile Hanoverian stallions, beginning -24, -12, 0, 12, 24 or 36 h after human chorionic gonadotrophin (hCG) administration. Mares were injected intravenously with 1500 IU hCG when they were in oestrus and had a pre-ovulatory follicle > or =40mm in diameter. Experiment 2 was a retrospective analysis of the breeding records of 2,637 mares inseminated in a total of 5,305 oestrous cycles during the 1999 breeding season. In Experiment 1, follicle development was monitored by transrectal ultrasonographic examination of the ovaries every 12 h until ovulation, and pregnancy detection was performed sonographically 16-18 days after ovulation. In Experiment 2, insemination data were analysed with respect to the number of live foals registered the following year. In Experiment 1, ovulation occurred within 48 h of hCG administration in 97.5% (391/401) of mares and the interval between hCG treatment and ovulation was significantly shorter in the second half of the breeding season (May-July) than in the first (March-April, P< or =0.05). Mares inseminated with cooled stallion semen once during an oestrus had pregnancy rates comparable to those attained in mares inseminated on two (48/85, 56.5%) or three (20/28, 71.4%) occasions at 24 h intervals, as long as insemination was performed between 24 h before and 12 h after ovulation (78/140, 55.7%). Similarly, a single frozen-thawed semen insemination between 12 h before (31/75, 41.3%) and 12 h after (24/48, 50%) ovulation produced similar pregnancy rates to those attained when mares were inseminated either two (31/62, 50%) or three (3/9, 33.3%) times at 24 h intervals.In the retrospective study (Experiment 2), mares inseminated with cooled semen only once per cycle had significantly lower per cycle foaling rates (507/1622, 31.2%) than mares inseminated two (791/1905, 41.5%), three (464/1064, 43.6%) or > or =4 times (314/714, 43.9%) in an oestrus (P< or =0.001). In addition, there was a tendency for per cycle foaling rates to increase when mares were inseminated daily (619/1374, 45.5%) rather than every other day (836/2004, 42.1%, P = 0.054) until ovulation.It is concluded that under conditions of frequent veterinary examination, a single insemination per cycle produces pregnancy rates as good as multiple insemination, as long as it is performed between 24 h before and 12 h after AI for cooled semen, or 12 h before and 12 h after AI for frozen-thawed semen. If frequent scanning is not possible, fertility appears to be optimised by repeating AI on a daily basis.     

Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen

Precise data on fertility results following peri- and postovulatory insemination in spontaneously ovulating gilts is lacking. Using transcutaneous sonography every 4 h during estrus as a tool for diagnosis of ovulation, the effects of different time intervals of insemination relative to ovulation were investigated with liquid semen (Experiment 1, n=76 gilts) and frozen semen (Experiment 2, n=80 gilts). In Experiment 3 (n=24 gilts) the number of Day-28 embryos related to the various intervals between insemination and ovulation was determined after the use of liquid semen. Using liquid semen the fertilization rates based on Day-2 to Day-5 embryos and the number of accessory spermatozoa decreased significantly in gilts inseminated with 2 x 10(9) spermatozoa per dosage in intervals of more than 12 h before or more than 4 h after ovulation. In the time interval 4 to 0 h before ovulation, comparable fertilization rates were obtained using frozen semen (88.1%) and liquid semen (92.5%). Fertilization rates and numbers of accessory spermatozoa decreased significantly when gilts were inseminated with frozen semen more than 4 h before or 0 to 4 h after the detection of ovulation. The percentage of Day-28 embryos was significantly higher following preovulatory insemination compared to inseminations 0 to 4 h and 4 to 8 h after ovulation. It is concluded that the optimal time of insemination using liquid semen is 12 to 0 h before ovulation, and 4 to 0 h before ovulation using frozen semen. The results stress the importance of further research on sperm transport and ovulation stimulating mechanisms, as well as studies on the time of ovulation relative to estrus-weaning intervals and estrus duration.     

Recovery rate and quality of embryos from mares inseminated after ovulation

The aim of this study was to evaluate the quality of embryos and their recovery rate from mares inseminated at different intervals after ovulation. Finnhorse and warmblood mares were inseminated with fresh semen 8 to 16 h, 16 to 24 h, or 24 to 32 h after ovulation. Control mares were inseminated before ovulation. Sixty-seven embryo flushings were performed between Days 7 and 9 after ovulation/insemination. Thirteen mares were not flushed, but their uteri were scanned for pregnancy on Days 14 to 16. Embryo recovery rates decreased as time from ovulation to insemination increased, although embryo quality remained normal as evaluated by morphological criteria and mitotic index. However, postovulatory insemination in this trial appeared to delay embryo development, since the embryos recovered from mares inseminated after ovulation were appreciably smaller and at an earlier stage of development than control embryos recovered from mares inseminated prior to ovulation. Part of this delay in embryo development in the postovulation group could be due to the time needed for sperm capacitation. In addition, as the time from ovulation to insemination increased, embryo development might have been further delayed by defects in the aging oocyte.     

Effect of timing of estradiol benzoate administration upon synchronization of ovulation in suckling Nelore cows (Bos indicus) treated with a progesterone-releasing intravaginal device

The present study investigated how the timing of the administration of estradiol benzoate (EB) impacted the synchronization of ovulation in fixed-time artificial insemination protocols of cattle. To accomplish this, two experiments were conducted, with EB injection occurring at different times: at withdrawal of the progesterone-releasing (P4) intravaginal device or 24h later. The effectiveness of these times was compared by examining ovarian follicular dynamics (Experiment 1, n=30) and conception rates (Experiment 2, n=504). In Experiment 1, follicular dynamics was performed in 30 Nelore cows (Bos indicus) allocated into two groups. On a random day of the estrous cycle (Day 0), both groups received 2mg of EB i.m. and a P4-releasing intravaginal device, which was removed on Day 8, when 400 IU of eCG and 150 microg of PGF were administered. The control group (G-EB9; n=15) received 1mg of EB on Day 9, while Group EB8 (G-EB8; n=15) received the same dose a day earlier. Ovarian ultrasonographic evaluations were performed every 8h after device removal until ovulation. The timing of EB administration (Day 8 compared with Day 9) did affect the interval between P4 device removal to ovulation (59.4+/-2.0 h compared with 69.3+/-1.7h) and maximum diameter of dominant (1.54+/-0.06 acm compared with 1.71+/-0.05 bcm, P=0.03) and ovulatory (1.46+/-0.05 acm compared with 1.58+/-0.04 bcm, P<0.01) follicles. In Experiment 2, 504 suckling cows received the same treatment described in Experiment 1, but insemination was performed as follows: Group EB8-AI48 h (G-EB8-AI48 h; n=119) and Group EB8-AI54 h (G-EB8-AI54 h; n=134) received 1mg of EB on Day 8 and FTAI was performed, respectively, 48 or 54 h after P4 device removal. Group EB9-AI48h (G-EB9-AI48 h; n=126) and Group EB9-AI54 h (G-EB9-AI54 h; n=125) received the same treatments and underwent the same FTAI protocols as G-EB8-AI48 h and G-EB8-AI54 h, respectively; however, EB was administered on Day 9. Conception rates were greater (P<0.05) in G-EB9-AI54 h [63.2% (79/125) a], G-EB9-AI48 h [58.7% (74/126) a] and G-EB8-AI48 h [58.8% (70/119) a] than in G-EB8-AI54 h [34.3% (46/134) b]. We concluded that when EB administration occurred at device withdrawal (D8), the interval to ovulation shortened and dominant and ovulatory follicle diameters decreased. Furthermore, when EB treatment was performed 24h after device removal, FTAI conducted at either 48 or 54 h resulted in similar conception rates. However, EB treatment on the same day as device withdrawal resulted in a lesser conception rate when FTAI was conducted 54 h after device removal.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).     

Fertility of swamp buffalo following the synchronization of ovulation by the sequential administration of GnRH and PGF2alpha combined with fixed-timed artificial insemination

This study evaluated fertility in swamp buffalo after synchronization of ovulation combined with fixed time artificial insemination. At the start of the study, designated day 0, from a group of 98 female Thai swamp buffalo, 55 buffalo (heifers n° = 20 and cows n° = 35) were selected to be synchronized with GnRH (Day 0) followed by PGF₂alpha (Day 7) and a second treatment with GnRH (Day 9). All buffalo were inseminated at two fixed times 12 h and 24 h after the second injection of GnRH (Ovsynch+TAI group); a second group of 43 buffalo (heifers n° = 19 and cows n° = 24) were not treated and were artificially inseminated (AI) at natural estrus (AI group). Blood samples were taken 22 days after insemination to evaluate progesterone plasma levels. In the Ovsynch+TAI group, overall conception rate (CR; i.e. the number of cows with progesterone >4.0 ng/ml on day 22 after AI divided by the number of animals inseminated), was 38.1% and overall pregnancy rate (PR; i.e. the number of cows that were pregnant at day 50-60 after insemination divided by the number of animals inseminated), was 32.7%. In the AI group overall CR and PR was 34.9%.Within the Ovsynch+TAI group, CR and PR were reduced (P < 0.05) in heifers compared with cows (CR 15.0% vs. 51.4% for heifers and cows, respectively; PR 15.0% vs. 42.9% for heifers and cows, respectively). Within the AI group the efficacy of treatment was similar between heifers and cows (CR and PR 31.6% for heifers and 37.5% for cows).In conclusion, this study indicates that in swamp buffalo it is possible to synchronize ovulation and use timed artificial insemination with the Ovsynch+TAI protocol.     

Evaluation of timed insemination during summer heat stress in lactating dairy cattle

We wished to compare the effect of summer heat stress on pregnancy rate in cows that were inseminated at a set interval associated with a synchronized ovulation vs those inseminated upon routine estrus detection. The study was carried out on a commercial dairy farm in Florida from May to September 1995. Lactating dairy cows were given PGF2 alpha (25 mg i.m.) at 30 + 3 d postpartum and randomly assigned to be inseminated at a set time (Timed group) or when estrus was detected (Control group). Cows in the Timed group were synchronized by sequential administration of Buserelin (8 micrograms i.m.) on Day 0 at 1600 h, PGF2 alpha (25 mg i.m.) on Day 7 at 1600 h and Buserelin (8 micrograms i.m.) on Day 9 at 1600 h. They were inseminated on Day 10 between 0800 and 0900 h (Day 9 + 16 h). Cows in the Control group were given PGF2 alpha at 57 + 3 d postpartum and inseminated when detected in estrus. Estrus detection or insemination rate for control insemination cows was 18.1 +/- 2.5% versus 100% for time inseminated cows (P < 0.01). Mean interval from PGF2 alpha to insemination was shorter for time inseminated cows (3 +/- 2.1 d < 35.5 +/- 1.9 d; P < 0.01). Pregnancy rate was greater for time inseminated cows (13.9 +/- 2.6 > 4.8 +/- 2.5%; P < 0.01) as was overall pregnancy rate by 120 d postpartum (27.0 +/- 3.6 > 16.5 +/- 3.5%; P < 0.05). Number of days open for cows conceiving by 120 d postpartum was less for time inseminated cows (77.6 +/- 3.8 < 90.0 +/- 4.2 d; P < 0.05), as was interval to first service (58.7 +/- 2.1 < 91.0 +/- 1.9 d; P < 0.01). Services per conception were greater for time inseminated cows (1.63 +/- 0.10 > 1.27 +/- 0.11; P < 0.05). The timed insemination program did improve group reproductive performance. However, the timed insemination program will not protect the embryo from temperature-induced embryonic mortality, but management limitations induced by heat stress on estrus detection are eliminated. An economical evaluation of the timed insemination program indicates an increase in net revenue per cow with implementation of timed insemination for first service during the summer months.     

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii)

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.     

Effect of time of ovulation and sperm concentration on fertilization rate in gilts

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.     

Methods of identifying and inseminating nonpregnant beef females after synchronization of second estrus with norgestomet implants

Beef females (547) were included in three experiments to evaluate methods of identifying and inseminating nonpregnant beef females after synchronization of second estrus with norgestomet implants. In the first experiment, heifers not pregnant to the first insemination were identified for insemination via estrus (inseminated via the a.m./p.m. rule or 48 h after implant removal). In the second experiment, females not pregnant to the first insemination were identified for insemination via estrus (inseminated via the a.m./p.m. rule) or progesterone concentrations < 1.5 ng/mL at implant removal (inseminated 48 h after implant removal). In the third experiment, heifers not pregnant to the first insemination were identified for insemination via progesterone concentrations (as in experiment 2) or anterior vagina electrical resistance values < 81 ohm resistance 48 h after implant removal (inseminated after resistance measured). All methods of identifying and inseminating nonpregnant females were equally effective (P > 0.10) and did not effect (P > 0.10) calving rates from the first and second AI.     

The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats.

Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25-50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25-33 h after hCG injection) and 30 were post-ovulatory (31-50 h after hCG injection). Pre-ovulatory females produced 10.5 +/- 1.1 follicles and no corpora lutea compared with 1.9 +/- 0.5 follicles and 7.5 +/- 0.9 corpora lutea for the post-ovulatory group (P < 0.05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2.8 +/- 1.5; P < 0.05) and embryos (0.4 +/- 0.3; P < 0.05) than post-ovulatory females (18.9 +/- 3.3 corpora lutea; 4.6 +/- 1.2 embryos). Two of the 14 cats (14.3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50.0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0.05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25-27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.     

Fertilization and ovum recovery rates in superovulated ewes following cervical insemination or laparoscopic intrauterine insemination at different times after progestagen withdrawal and in one or both uterine horns

In Exp. 1, 40 ewes were used in a 2 x 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P less than 0.01) decrease in egg recovery of over 40% compared with cervically inseminated ewes. In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria. In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P less than 0.05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery. From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.     

Effects of insemination-ovulation interval on fertilization rates and embryo characteristics in dairy cattle

The objective of this study was to examine effects of the interval between insemination and ovulation on fertilization and embryo characteristics (quality scored as good, fair, poor and degenerate; morphology; number of cell cycles and accessory sperm number) in dairy cattle. Time of ovulation was assessed by ultrasonography (every 4h). Cows were artificially inseminated once between 36h before ovulation and 12h after ovulation. In total 122 oocytes/embryos were recovered 7d after ovulation. Insemination-ovulation interval (12h-intervals) affected fertilization and the percentages of good embryos. Fertilization rates were higher when AI was performed between 36-24 and 24-12h before ovulation (85% and 82%) compared to AI after ovulation (56%). AI between 24 and 12h before ovulation resulted in higher percentages of good embryos (68%) compared to AI after ovulation (6%). Insemination-ovulation interval had no effect on number of accessory sperm cells and number of cell cycles when corrected for embryo quality. This study showed that the insemination-ovulation interval with a high probability of fertilization is quite long (from 36 to 12h before ovulation). However, the insemination-ovulation interval in which this fertilized oocyte has a high probability of developing into a good embryo is shorter (24-12h before ovulation).     

Low dose insemination in synchronized gilts

Conventional insemination techniques in pigs require 2 to 3 x 10(9) sperm/dose. When using the latest high-speed sperm-sorting technology, one can still sort only about 5 to 6 million sperm of each sex per hour. The objective of the present study was to find the minimal sperm concentration at a low-insemination volume in pigs without diminishing fertilization rate and litter size using surgical deep intra-uterine insemination (IUI). Semen from 3 boars was collected and diluted with Androhep to 5 x 10(8), 1 x 10(8), 1 x 10(7), 5 x 10(6) or 1 x 10(6) sperm/0.5 ml. In trial 1, 109 prepuberal gilts were synchronized and surgically inseminated into the tip of each uterine horn 32 h or 38 h after hCG treatment or at the time of ovulation, respectively. Pregnant gilts were allowed to go to term. Pregnancy and farrowing rates did not differ significantly except at the lowest sperm concentration if inseminated 32 h or 38 h after hCG treatment (p < 0.05). No differences were found among insemination groups for the total number of piglets, number of piglets born alive, stillborn piglets, and mummified fetuses. In trial 2, 34 gilts were inseminated as described above 32 h after hCG. Additionally, 9 gilts were inseminated once nonsurgically with 1 x 10(9) sperm as controls. Gilts were slaughtered 48 h after insemination, and embryos were recovered. Embryos were cultured in NCSU 23 (120 h), evaluated morphologically and stained with fluorescent dye (Hoechst 33342) to visualize nuclei. Recovery rates varied between 71.4% and 84.4%. Fertilization rate of the lowest sperm concentration (1 x 10(6) sperm/horn) differed significantly (p < 0.05) from all other groups. Cleavage rates at specific developmental stages did not differ. After 5 days of in vitro culture, embryos developed to morulae and blastocysts. No differences were found for these stages. In conclusion, no major differences were found between insemination groups as long as the sperm dosage was at least 10 million sperm per gilt. The low volume was sufficient for successful deep intra-uterine insemination. Embryo development was comparable to the controls.     

Intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: use of an infusion pipette and the Osiris catheter

One hundred fifty-two bitches of seven breeds were vaginally inseminated with fresh or frozen-thawed semen of 10 stud dogs of respective breeds. The semen was supplemented with prostatic fluid before insemination. In experiment 1 bitches of each breed were randomly assigned to three treatment groups, consisting of 29 females (group 1), 33 females (group 2) and 32 females (group 3). In group 1 bitches were inseminated into vagina with fresh semen using a bovine infusion pipette. In group 2 bitches were inseminated into vagina with fresh semen using the Osiris catheter. In group 3 bitches were inseminated with frozen-thawed semen with the Osiris catheter. The number of sperms in each insemination dose was adjusted to 300 x 10(6). In experiment two bitches were randomly assigned to two treatment groups, consisting of 30 females (group A) and 28 females (group B). In group A bitches were inseminated with fresh semen, whereas in group B with frozen-thawed semen. Osiris catheter was used in both groups. The total number of sperms was adjusted to provide 250 x 10(6) of progressively motile spermatozoa in each insemination dose. In experiment 1 the pregnancy rates/whelping rates were 86.2/82.8%, 81.8/81.8% and 59.4/59.4% for groups 1, 2 and 3, respectively. The differences between group 1 and 3 were statistically significant (p < 0.05). The litter sizes at birth/litter sizes at weaning were 5.8+/-2.3/5.4+/-2.0, 6.3+/-1.4/5.7+/-1.0 and 3.9+/-1.2/3.5+/-1.5 in groups 1, 2 and 3, respectively. The litter size at birth and at weaning was reduced (p < 0.05) when frozen-thawed semen was used for insemination (group 3). There were not significant (p > 0.05) differences in the litter size between groups 1 and 2. In experiment 2 pregnancy rates/whelping rates and litter sizes at birth/litter sizes at weaning were 86.7/86.7%, 60.7/57.1% (p < 0.05) and 6.1+/-1.6/5.7+/-1.7, 4.0+/-1.4/3.8+/-1.4 (p < 0.05) in groups A and B, respectively. This study shows that results of AI with a fresh semen using a bovine infusion pipette and the Osiris catheter are equivalent. The results of the use of the Osiris catheter for vaginal insemination of frozen-thawed dog semen extended with prostatic fluid after thawing are not encouraging. The pregnancy rate, whelping rate and litter size are reduced when frozen-thawed, prostatic fluid-supplemented semen is vaginally deposited using the Osiris catheter.