Embryo production from superovulated cattle following insemination of sexed sperm

Two trials were conducted to ascertain fertilization rate, embryo quality and numbers of transferable embryos in superovulated heifers and cows inseminated with sexed sperm. Inseminates contained 2 x 10(6), 10 x 10(6) or 20 x 10(6) total sperm enriched for the X- or Y-chromosome ( approximately 90%) by flow cytometry/cell sorting. Non-sexed inseminates contained 40 x 10(6) total sperm. Donors in each trial were allocated to one of each of the bulls included in that study. Each donor was inseminated with frozen/thawed sperm from the same bull for each treatment in successive courses of superstimulation with twice daily i.m. injections of FSH for 4 d. Heifers and cows were inseminated 12 and 24 h after visually observed standing estrus in Trial 1. In Trial 2, a single timed inseminate was used 70-72 h following PGF(2alpha). Ova/embryos were collected non-surgically 7-7.5 d after insemination. In both trials, fewer ova were fertilized with sexed versus non-sexed treatments and with 2 x 10(6) sexed sperm compared to higher doses (P < 0.05). However, insemination of 20 x 10(6) total sexed sperm of >or=90% purity resulted in similar numbers of transferable embryos of the desired sex compared to that for non-sexed sperm.     

Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers

This experiment was designed to test whether spermatozoa encapsulated in an alginate poly-L-lysine matrix had an extended fertile life in vivo after insemination. Estrus was synchronized in 417 primiparous Friesian and Jersey heifers with a system based on a CIDR-B intravaginal device before the heifers were inseminated either during proestrus (24 h after device removal) or at estrus (48 h after device removal). Pregnancy rates to first inseminations did not differ between the 24 and 48 h inseminations (61 vs 60.6%) with liquid semen diluted in Caprogen (control) but differed with encapsulated semen (45.1 vs 68.6%). The difference in pregnancy rates between the 2 types of semen was more pronounced (P < 0.08) in the animals that were visually detected in estrus. The mean survival time of spermatozoa in the female reproductive tract following insemination at the 24-h insemination time was estimated to be 50 +/- 7.5 h. The increased pregnancy rate with insemination of encapsulated spermatozoa at 48 h could have been due to this process predisposing spermatozoa to capacitate soon after insemination.     

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.     

Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa

The present study evaluates the effectiveness of the transcervical deep intrauterine insemination (DUI) with a reduced number of frozen-thawed boar spermatozoa in weaned sows. DUI was performed using a specially designed flexible device (length 180 cm, outer diameter 4mm, working channel 1.8mm, working channel's volume 1.5 ml) that was inserted through an artificial insemination spirette to cross the cervix lumen and moved into one uterine horn as far as possible. Spermatozoa diluted in 7.5 ml of BTS were flushed into the uterine horn by a syringe attached to the working channel. In Experiment 1, 111 hormonally treated (eCG/hCG) weaned sows were inseminated once using one of the following three regimens: (1) DUI with frozen-thawed spermatozoa (1000 x 10(6) cells per dose; n=49); (2) DUI with fresh semen (150 x 10(6) cells per dose; n=29, as control of DUI procedure); and (3) cervical insemination with frozen-thawed spermatozoa (6000 x 10(6) cells diluted in 100ml; n=33). No differences (P>0.05) were found for farrowing rates (77.55, 82.76, and 75.76, respectively) or litter sizes (9.31+/-0.41, 9.96+/-0.32, and 9.60+/-0.53 piglets born per litter, respectively) among the groups. In Experiment 2, DUI was performed on the spontaneous estrus in weaned sows (2-6 parity) with 1000 x 10(6) frozen-thawed (40 sows) or 150 x 10(6) fresh spermatozoa (38 sows). The farrowing rate of sows inseminated twice with frozen-thawed spermatozoa (70%) was significantly (P<0.05) lower than with fresh semen (84.21%). No significant difference (P>0.05) was found in litter size between frozen-thawed spermatozoa (9.25+/-0.23 piglets born per litter) and fresh semen (9.88+/-0.21 piglets born per litter). These preliminary results indicate that application of DUI provides acceptable fertility in weaned sows using a relatively low number of frozen-thawed spermatozoa.     

Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled, and frozen stallion semen

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1 x 10(9) fresh spermatozoa, or into the oviduct with 2 x 10(5) fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 degrees C before intrauterine insemination with 2 x 10(9) spermatozoa or intraoviductal inseminations of 2 x 10(5) spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.     

Birth of piglets after deep intrauterine insemination with flow cytometrically sorted boar spermatozoa

The present study was carried out to determine the pregnancy rates, farrowing rates and litter size in sows with either induced or spontaneous ovulation inseminated with flow cytometric sorted spermatozoa using deep intrauterine insemination technology. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry/cell sorting but not separated into separate X and Y populations. In Experiment 1, sows (n=200) were weaned and treated for estrus/ovulation induction with eCG/hCG. Inseminations with either sorted (70 or 140 million) or non-sorted (70 or 140 million) spermatozoa were done using a specially designed flexible catheter. Farrowing rates were 39.1 and 78.7% for 70 million of sorted and non-sorted, respectively, and 46.6 and 85.7% for 140 million of sorted and non-sorted, respectively (P<0.05). The litter size in sows inseminated with sorted spermatozoa showed a tendency to be lower than when non-sorted spermatozoa were inseminated. In Experiment 2, sows (n=140) were inseminated as in Experiment 1 except that natural estrus was used. The ovaries of these sows were evaluated by transrectal ultrasonography. Farrowing rates were 25 and 77.2% for 70 million of sorted and non-sorted, respectively, and 32 and 80.9% for 140 million of sorted and non-sorted, respectively (P<0.05). These results show that the Deep Intrauterine Insemination technology can be successfully used to produce piglets from sorted spermatozoa when sows are hormonally treated to induce synchronous post weaning oestrus and ovulation.     

Early pregnancy loss in sows after low dose, deep uterine artificial insemination with sex-sorted, frozen-thawed sperm

Recent developments in reproductive technologies have enabled the production of piglets of a predetermined sex via non-surgical, low dose artificial insemination. The practical application of sex-sorting technology to the pig is made challenging by the large numbers of sperm required for successful insemination of sows. One way of overcoming the time required for sex-sorting may be to create a bank of cryopreserved, sex-sorted sperm, thus making available appropriate doses as sows require insemination. To date, little success has been achieved with non-surgical inseminations of sex-sorted boar sperm. This study attempted to achieve litters of a predetermined sex after a double insemination of sows with 160x10(6) sex-sorted, frozen-thawed sperm. Sows were synchronised and sperm were non-surgically inseminated into the proximal third of the uterine horn at 36 and 42 h after hCG administration. Sows inseminated with sex-sorted sperm achieved similar pregnancy rates to those receiving an equal dose of unsorted, frozen-thawed sperm. However, all sows conceiving after insemination with sex-sorted sperm returned to oestrus within 57 days of insemination. This was a higher rate of pregnancy loss than observed for sows inseminated with unsorted sperm (37.5%; P=0.031). A combination of low sperm numbers and potentially compromised developmental capability of embryos derived from sex-sorted sperm may have resulted in this early stage loss of pregnancy.     

Analysis of bovine sexed sperm for IVF from sorting to the embryo

The objective of this study was to characterize bovine semen parameters and determine the best IVF conditions to produce a maximal percentage of blastocysts. Four types of semen were analyzed with CASA and flow cytometry: fresh and frozen non-sexed semen; fresh and frozen sexed semen. Semen was obtained from four Holstein bulls and two ejaculates from each bull were analyzed. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro with all types of semen (for sexed semen, 2, 5 or 10 μg/mL heparin was added to the IVF media while for non-sexed semen, 10 μg/mL was added in the IVF medium). Presumptive zygotes were co-cultured with Buffalo rat liver cells in Menezo's B2 medium, and cleavage rates at Day 2, and blastocyst rates at Day 7 of culture, were recorded. Sexed semen resulted in fewer blastocysts than non-sexed semen (P < 0.05), and certain bulls performed better in IVF. Freezing, and not sexing, had a more significant negative effect on semen quality. Compromised semen quality due to sexing and/or freezing can explain the reduced in vitro blastocyst rates when using frozen-thawed sexed semen. Sexed semen that appeared more capacitated seemed to require less heparin in IVF than sexed semen that appeared less capacitated to produce a maximal percentage of blastocyst. Flow cytometry sorting eliminates spermatozoa that possess compromised DNA, and therefore the reduced fertility seen in vitro is not due to an increased percentage of spermatozoa with compromised DNA. This study describes tools that can monitor semen parameters to optimize IVF conditions and thus obtain maximal blastocyst rates.     

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.     

Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste

Twenty-five bitches were artificially inseminated with semen that was frozen-thawed using an egg yolk-Tris-glucose-citrate extender containing 5% glycerol with, or without the addition of 0.5% Equex STM Paste. Semen was collected on 2 occasions from 11 dogs, pooled, and evaluated for sperm motility, morphology and plasma membrane integrity. Each pool was then divided in 2 parts, diluted with 1 of the 2 extenders, and frozen in 0.5-mL straws. In the bitches, plasma progesterone was assayed daily during late proestrus and estrus. Artificial insemination (AI) was performed twice on Days 3 and 5 after the estimated LH peak. For each insemination, 200x10(6) spermatozoa were used. Ten bitches were inseminated with semen frozen without Equex: In 5 females, semen was deposited transcervically into the uterus with the aid of a fiberoptic endoscope and a urethral catheter, while the remaining 5 bitches were inseminated in the cranial vagina using a Norwegian catheter. Fifteen bitches were inseminated with semen frozen-thawed with Equex: Two groups of 5 bitches were inseminated according to the techniques described above, while 5 bitches were inseminated vaginally using the Osiris catheter. Pregnancy was diagnosed and the number of fetuses counted by ultrasound examination. Post-thaw, spermatozoa frozen with Equex tended to have higher total and progressive motility and to survive longer in vitro than when the extender without Equex was used. Spermatozoal concentration, age of the bitches, duration of heat and estrus, and progesterone concentration at LH peak and at the first and second AI did not differ among the 5 groups. The overall pregnancy rate of 84% (21/25) was close to what can be expected from well controlled natural matings. For both freezing extenders tested, 5/5 bitches were pregnant after uterine deposition of semen and 4/5 were pregnant when semen was deposited in the anterior vagina using the Norwegian catheter. With the Osiris catheter, 3/5 inseminations resulted in a pregnancy. No significant differences in pregnancy rate or number of fetuses were found between groups, site of deposition or freezing extender.     

Effect of exogenous gonadotropins on the weaning-to-estrus interval in sows

The efficacy of PG 600 (400 IU PMSG and 200 IU hCG) for accelerating the onset of estrus was determined for sows weaned during the summer. Yorkshire sows (average parity = 4.6), nursing 8.6 +/- 0.2 pigs (mean +/- SEM) were weaned after 27.7 +/- 0.4 d of lactation and were treated intramuscularly with either PG 600 (n = 35) or with 0.9% saline (n = 35). Sows were checked for estrus once daily in the presence of a mature boar. Treatment with PG 600 increased (P < 0.05) the percentage of sows in estrus within 7 d after weaning (97.1 vs 82.9%). Relative to controls, sows given PG 600 expressed estrus sooner (3.8 +/- 0.1 d vs 4.5 +/- 0.1 d; P < 0.01). Sows exhibiting estrus within 7 d after treatments were artificially inseminated 0 and 24 h after first exhibiting estrus. The percentage of inseminated sows that farrowed tended to be higher (P < 0.07) for control than for PG 600-treated sows (96.6 vs 82.3%). The number of pigs born live was similar (P > 0.1) for sows treated with PG 600 and with saline, and was 12.7 +/- 0.6 and 11.7 +/- 0.7, respectively. Pigs farrowed by saline-treated sows, however, tended to be heavier (P < 0.09) than pigs farrowed by sows treated with PG 600 (1.49 +/- 0.06 kg vs 1.34 +/- 0.06 kg). In summary, PG 600 accelerated the onset of estrus in sows weaned during the summer. Sows mated during the induced estrus, however, tended to have a lower farrowing rate and farrowed lighter pigs than control sows inseminated during a natural estrus occurring within 7 d after weaning.     

Influence of time of insemination relative to ovulation and frequency of insemination on gilt fertility

The objectives of this study were to determine the optimal time of insemination in the pre-ovulatory period (from 32 to 0 h before ovulation) and to evaluate once-daily versus twice-daily inseminations in gilts. In Experiment 1, pre-puberal gilts (n=102) were observed for estrus every 8h and ultrasonography was performed every 8h from the onset of estrus to confirmation of ovulation. The gilts were inseminated once with 4 x 10(9) spermatozoa at various intervals prior to ovulation. Pregnancy detection was conducted 24 days after AI and gilts were slaughtered 4-6 days later. Corpora lutea and the number of viable embryos were counted and the embryo recovery rate was calculated (based on the percentage of corpora lutea). Inseminations performed <24h before ovulation resulted in a higher embryo recovery rate (P=0.02) and produced 2.1 more embryos (P=0.01) than inseminations >or=24h before ovulation. However, the pregnancy rate was reduced when inseminations were performed >16 h before ovulation (P=0.08). In Experiment 2, pre-puberal gilts (n=105) were observed for estrus every 12h and ultrasonography was performed every 12h from the onset of estrus to confirmation of ovulation. Gilts were inseminated (with 4 x 10(9) spermatozoa) 12h after the onset of estrus, with inseminations repeated either every 12h (twice-daily) or 24h (once-daily) during estrus. The gilts were allowed to farrow. There were no differences (between gilts bred twice-daily versus once-daily) for return to estrus rate (P=0.36) and adjusted farrowing rate (P=0.19). However, gilts inseminated once-daily had 1.2 piglets less than those inseminated twice-daily (P=0.09). In conclusion, gilts should be inseminated up to 16 h before ovulation, as intervals >16 h reduced pregnancy rate and litter size.     

Semen backflow after insemination and its effect on fertilisation results in sows

The aim of the present study was to investigate the volume of and number of spermatozoa in semen backflow during and after insemination, and the effect of backflow on fertilisation results assessed at day 5 of pregnancy. Multiparous sows (n=140) were artificially inseminated with either (1, 3 or 6)×10⁹ mixed spermatozoa from three boars in a constant volume of 80 ml. Backflow of semen was measured three times: during insemination (M1); during the first half hour after insemination (M2); and from 0.5 h until about 2.5 h after insemination (M3). Transrectal ultrasonography was performed at intervals of 4 h to determine the time of ovulation. Sows were sacrificed at 120±0.4 h after ovulation to assess the results of fertilisation. Every sow had some backflow and the variation in volume, and number of spermatozoa within the backflow was high. The average semen backflow within 2.5 h after insemination was 70±3.4% of the volume and 25±1.4% of the spermatozoa of the inseminated dosage. The concentration of the backflow (% of the inseminated dosage) decreased with time after insemination from 65% at M1 to 40% and 26% at M2 and M3, respectively. The correlations between volume and number of spermatozoa were high: r=0.97, r=0.73 and r=0.81 in M1, M2 and M3, respectively. More than 5% of the inseminated spermatozoa in backflow during insemination affected fertilisation negatively in those sows inseminated with 1×10⁹ spermatozoa (P<0.05). Backflow after insemination had no effect on fertilisation results (P>0.05). Timing of insemination relative to ovulation and oestrus were not related to backflow during or after insemination (P>0.05). Of the sows which had backflow, those of parity 1 tended to have the highest proportion of sows with more than 5 ml backflow (47%; n=8 of 17) compared with sows from parity 2 and higher (24%; n=14 of 59) (P=0.075). It was concluded that excessive backflow of semen during insemination had a negative effect on fertilisation results when sows where inseminated with only 1×10⁹ spermatozoa. Causes of variation in backflow between sows were not clearly identifiable.     

Fertility of frozen-thawed stallion semen extended in lactose-EDTA-egg yolk extender and packaged in 1.0-ml straws

The fertility of frozen-thawed and fresh semen from each of three stallions was compared in an experiment with a randomized block design using 128 mares. Semen was collected every third day, extended in lactose-EDTA-egg yolk extender at a concentration of 500 × 10⁶ progressively motile sperm per 1.0 ml, and frozen in individual-dose, 1.0-ml straws (1.9 mm × 267 mm). The same stallions were collected daily for inseminations with fresh semen. For each insemination dose with fresh semen, 300 × 10⁶ progressively motile sperm were added to 10 ml of heated skim milk extender. Mares were inseminated daily from the second day of estrus through the end of estrus. Of 52 ejaculates processed and frozen, 38% were discarded because < 35% of the sperm were progressively motile after thawing. Based on rectal palpations on day 50 post-ovulation, pregnancy rates for inseminations during one estrus to semen from the three stallions were 17, 33 and 35% for frozen-thawed semen and 60, 62 and 64% for fresh semen. Pregnancy rates with frozen semen from two of the three stallions were 54% of the rates attained with fresh semen.     

Fertility of fresh and frozen-thawed goat semen during the nonbreeding season

A total of 4109 does of a local Greek breed (Capra prisca) were synchronized with intravaginal MPA-sponges and PMSG, and 24 bucks of Alpine (n = 8), Saanen (n = 8) and Damascus (n = 8) breeds were used for studying the fertility of nonfrozen and frozen-thawed semen during the nonbreeding season (June to August). Artificial insemination (AI) was performed once (50 to 55 h after sponge withdrawal) or twice (36 and 60 h after sponge withdrawals with fresh semen (collected during the nonbreeding season, stored at 16 degrees C and inseminated within 6 h) or frozen semen (prepared from the same bucks during the preceding breeding season). The induction of estrus was successful, varying between 91.0 and 95.0%. The form of semen (fresh or frozen-thawed used for inseminating the synchronized does affected their fertility: the overall kidding rate with fresh semen (65.5%) was higher (P < 0.05) than that with frozen-thawed semen (53.4%). The fertility level was also affected by the number of inseminations performed: the overall kidding rate was significantly higher (P < 0.001) in the does inseminated twice with fresh or frozen-thawed semen (70.4 and 59. 1%, respectively) than in those inseminated only once (48.9 and 44.9%, respectively). Finally, the breed of the buck used for preparing the fresh or the frozen-thawed semen affected the fertility level of the does. The kidding rate was higher in does inseminated with fresh semen prepared from bucks of the Damascus breed than from bucks of Saanen or Alpine breed. However, when frozen-thawed semen was used the kidding rate was lower in does inseminated with semen prepared from bucks of the Damascus breed than from bucks of the Alpine or Saanen breed. It is concluded that the fresh semen of Alpine, Saanen and Damascus breed bucks, born and raised under the climate conditions prevailing in Greece (34 degrees to 41 degrees N), can be used successfully during the nonbreeding season (June to August) for inseminating does.     

Fertilization rates and in vitro embryo production using sexed or non-sexed semen selected with a silane-coated silica colloid or Percoll

The aim of this study was to evaluate sperm fertilization rates and in vitro embryo development rates for sexed and non-sexed semen selected using a silane-coated silica colloid method (Isolate) or Percoll. Frozen/thawed, sexed and unsexed semen samples from four Holstein bulls were randomly allocated to one of two different density gradient selection methods. Sperm quality (motility, concentration, morphology and membrane integrity) were evaluated and compared before and after sperm selection. Sperm motility and morphology improved (P < 0.005) after the sperm selection process with no differences between the two methods. For non-sexed semen, Percoll gradient increased the mean (± SEM) percentage of sperm recovered (57.3 ± 2.8) compared to Isolate (46.0 ± 1.8; P < 0.01). However, membrane integrity was higher after Isolate than Percoll (sexed semen: 41.0 ± 0.6 vs. 38.8 ± 0.8 and non-sexed semen 60.8 ± 1.6 vs. 58.8 ± 0.5; P < 0.05). The percentage of blastocysts produced was higher when either sexed or non-sexed semen was selected by Isolate (14.0 ± 1.0; 22.0 ± 1.1) than by Percoll (10.5 ± 1.5; 17.0 ± 2.1, respectively; P < 0.05). In summary, Isolate was a more effective method for the recovery of high quality sperm for in vitro fertilization embryo production.     

Fertility of frozen-thawed porcine semen following controlled-rate freezing in straws

A method was developed for freezing large batches of porcine semen in straws at a controlled rate in a liquid nitrogen programmable freezer. The fertilizing potential of spermatozoa frozen by this method was examined by inseminating 220 sows with a mixture of semen from two boars. Estrus was synchronized using one of two regimens and sows were inseminated once at 34 h after human chorionic gonadotropin (hCG) treatment. The average pregnancy rate at 60 d of gestation, farrowing rate and litter size were 60.9%, 51.4% and 8.8, respectively. The fertilizing potential of spermatozoa frozen by this method appeared to be similar to that reported for other methods of freezing porcine semen.     

A method of artificial insemination in captive White-tailed deer (Odocoileus virginianus )

Production of fawns by artificial insemination in captive White-tailed deer (Odocoileus virginianus ) has been accomplished by using frozen-thawed spermatozoa. The purpose of this study was to determine if frozen-thawed semen deposited at the posterior face of the os cervix could produce conception. Five hand-raised female White-tailed deer and one hand-raised male White-tailed deer were used over two breeding seasons 1984-1985 and 1985-1986. The vasectomized buck was ued to detect estrus in the does. The does were inseminated with frozen-thawed semen containing at least 100 million live normal cells with a 60% or higher motility. The artificial insemination catheters used in this study worked well, but due to the small size of the cervix, the catheter could only be passed up to the first cervical ring, the site at which the semen was deposited. Over two breeding seasons, nine does were inseminated with frozen-thawed spermatozoa; each doe was inseminated once each estrous cycle at one of the following times: 0, 6, 12, 18, 24 or 30 h. after detection of estrus. Of the nine does inseminated with frozen-thawed spermatozoa, six conceived and carried to term 11 healthy normal fawns, yielding an overall conception rate of 67%.     

Embryo production in superovulated Angus cows inseminated four times with sexed-sorted or conventional, frozen-thawed semen

This study tested the hypothesis that four inseminations of commercially frozen sexed semen (≥2.1 × 10⁶ sperm per 0.25-mL straw) in superstimulated embryo donors would yield a percentage and quantity of transferable embryos similar to that achieved with conventional frozen semen. Bos taurus, angus cows (n = 32), stratified by age and body condition, were randomly allocated to receive four inseminations of frozen-thawed semen, either conventional semen (≥15 × 10⁶ sperm/straw; Conventional) or sexed semen (≥2.1 × 10⁶ sperm/straw; Sexed) from one of two AI sires. From 10 to 13 d after estrus, follicle-stimulating hormone (FSH) was given twice-daily, with prostaglandin F_2α given twice on the last day. Cows were inseminated once (1×) at first detected estrus and twice (2×) and once (1×) at 12 and 24 h later, respectively, with nonsurgical embryo recovery 7 d after first detected estrus. The study was repeated 30 d later (switch-back experimental design). The total number of ova per flush was similar between Conventional and Sexed treatments (10.9 ± 1.8 vs. 10.5 ± 1.6), but the number of Grade 1 embryos was greater (P < 0.01) for Conventional (4.3 ± 0.8 vs. 2.3 ± 0.7). Conversely, the mean number of unfertilized ova was greater (P < 0.05) for Sexed (5.6 ± 1.0 vs. 3.0 ± 1.2). There was no significant difference between treatments for numbers of degenerate, Grades 2 or 3, and transferable embryos and no significant differences between bulls in percentage of transferable embryos (44.4% and 46.7%). However, fertilization rates and percentage of transferable embryos were affected (P < 0.05) by period and donor. In conclusion, superstimulated donor cows inseminated four times had fewer Grade 1 embryos and more unfertilized ova with sexed versus conventional semen.     

Does intrauterine site of insemination in cattle really matter?

Two trials were conducted to determine the influence of semen placement on pregnancy rate in dairy heifers and cows. Seventy-two dairy heifers were artificially inseminated (AI) 10 to 12 h after the first detection of estrus. Control heifers (n = 25) were inseminated at the junction of the uterine body and internal cervical os. The remaining heifers were inseminated deep in one uterine horn, 3 to 5 cm anterior to the external bifurcation. Twenty-three heifers were inseminated in the horn ipsilateral to the ovary bearing the ovulatory follicle, and 24 heifers were inseminated in the contralateral horn. Pregnancy rates did not differ for the three groups of heifers. In a second trial, 64 inseminations were performed in 38 nonlactating, adult dairy cattle. Thirty-one inseminations were made deep in the uterine horn ipsilateral to the ovary bearing the ovulatory follicle and 33 in the contralateral horn. Pregnancy rates were similar for both groups. Combining both trials, pregnancy rates for ipsilateral and contralateral inseminations were equal (32 54 = 59% and 34 57 = 60% , respectively). Therefore, placement of semen in one horn of the uterus does not appear to be a cause of decreased or increased pregnancy rate with AI.