Effect of frozen semen on the uterus of mares with pathological uterine changes

Pregnancy rates after frozen semen inseminations (AI), particularly in older and problem mares, are lower than after fresh semen AI. Uterine contractility and the inflammatory reaction after frozen semen insemination were studied in two groups of mares: the abnormal group comprised of 6 old barren mares categorized in biopsy category IIB or III, and the control group including 6 reproductively normal young maiden mares in biopsy category I or IIA. All 12 mares were inseminated in the first cycle with 2 mL of phosphate-buffered saline (PBS) and in their second cycle with 2 mL of frozen semen containing 800 x 10(6) spermatozoa. Before and 1, 2, 4, 8, and 20 to 24 h after this treatment, all mares were examined by ultrasonography for intrauterine fluid accumulations (IUFA). The examinations were videotaped to count the number of uterine contractions later. Uterine fluid was obtained by tampon before treatment, and by the tampon method followed by uterine lavage after the last examination. Fluids were cultured bacteriologically, and polymorphonuclear leukocytes (PMN) were counted. Trypsin-inhibitor capacity (TIC), lysozyme concentration, and beta-glucuronidase (BGase) and N-acetyl-beta-D-glucosaminidase (NAGase) activities were determined in frozen-thawed tampon and lavage fluids. Both treatments induced significant neutrophilia in the uterine lumen. Although PMN concentrations were numerically higher after frozen semen AI than after PBS-treatment, the difference was not significant. There was not any difference between the mare groups either. The amount of IUFA differed only in the normal group between frozen semen AI and PBS treatment, and between 0- and 24-h samples for frozen semen AI. Although abnormal mares showed consistently more fluid than normal mares, this difference was not significant. Uterine contractions and enzyme concentrations between groups did not differ. None of the variables showed significant differences between the normal and abnormal mares in their reaction to frozen semen AI.     

Successful uterine insemination of fallow deer with fresh and frozen semen

Six fallow does were inseminated directly into the uterine horns 72 h (three does) or 78 h (three does) after the removal of progestagen intravaginal sponges. Three does were inseminated with fresh (two at 72 h and one at 78 h) or frozen-thawed (one at 72 h and two at 78 h) semen. The semen used had been collected by electroejaculation and had been stored for 2 yr in liquid nitrogen in a Tris, citric acid, glycerol diluent containing 2.25% egg yolk. Three does each produced a live fawn to insemination and all does had been inseminated 72 h after removal of sponges; two with fresh semen and one with frozen semen. The remaining three does failed to conceive to insemination, but did produce fawns to mating at a subsequent estrus.     

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.     

Effect of insemination dose and site on uterine inflammatory response of mares

It is unclear whether AI of mares deep into the uterine horn causes more or less inflammation of the endometrium than conventional AI. Thus, we compared uterine inflammatory reactions of mares inseminated with two different doses of frozen-thawed semen into the tip of the uterine horn (UH) ipsilateral to the preovulatory follicle with those of mares inseminated into the uterine body (UB). Thirty-two mares were assigned to one of four groups (eight mares/group): UB20=AI into UB, 20 x 10(6)sperm/0.5 mL; UB200=AI into UB, 200 x 10(6)sperm/0.5 mL; UH20=AI into UH, 20 x 10(6)sperm/0.5 mL; UH200=AI into UH, 200 x 10(6)sperm/0.5 mL, and inseminated 24 h after hCG administration. Before and 24 h after AI, they were examined with ultrasonography for the presence of intrauterine fluid. At 24 h, uterine fluid samples were obtained first by absorbing fluid into a tampon and then by uterine lavage. Uterine fluid was examined for polymorphonuclear leukocytes (PMN) and bacteriology, and frozen for lysozyme and TIC (trypsin-inhibitor capacity) assays. Only three mares conceived, one in each of the following groups: UB200, UH20, and UH200. Mares in the UH20 group accumulated less intrauterine fluid (p<0.05) than those in the other groups, which had similar amounts. No significant differences in PMN numbers were detected in either tampon or lavage fluid. Enzyme levels between groups did not differ statistically, except for TIC, which was lowest in the UH200 group. Thus, deep uterine horn AI caused no greater inflammation or irritation than uterine body AI in normal mares 24 h after insemination.     

Effect of oxytocin and flunixin meglumine on uterine response to insemination in mares

The most probable reason for persistent postbreeding endometritis in mares is weak myometrial contractility. The influence of oxytocin (OT; an ecbolic agent) and flunixin meglumine (FLU; a prostaglandin inhibitor serving as a model for mares with decreased uterine contractility) on uterine response to artificial insemination (AI) was studied in mares with no history of reproductive failure. The mares were treated intravenously with 10 mL saline (Group C, n = 10) or 0.01 IU/kg OT (Group OT, n = 10) 2, 4, 8, and 25 h after AI. Group FLU (n = 11) was treated with 1.1 mg/kg FLU 2 h after AI and with saline thereafter. The mares received the same treatments in the first and third cycles but were sampled either at 8 or 25 h. The amount of intrauterine fluid (IUF) and edema and the number of uterine contractions were recorded before AI and 10 min after the treatments using transrectal ultrasonography. At 8 h after AI, the mares were treated with human chorionic gonadotropin, and, after 8-h or 25-h scans, a 500-mL uterine lavage and a biopsy were performed. Ovulation was confirmed at 48 h and pregnancy 14 to 17 d after AI. No manipulations were done during the second estrus. At 8 h after AI, Group FLU had more polymorphonuclear leukocytes (PMNs) in the uterine lavage fluid than did Group OT (P < 0.05), but uterine contractions did not differ significantly. At 25 h, the PMN concentrations were low in all groups. Group OT rarely showed IUF. The uterine biopsy specimens of Group FLU showed less inflammation of the stroma but more PMNs in the uterine lumen 8 h after AI than that of the control group (P < 0.05). The pregnancy rates did not differ between the groups (63% C, 53% OT, and 50% FLU). Oxytocin rapidly and effectively removed IUF and PMNs after AI and thereby shortened the duration of postbreeding inflammation.     

Therapeutic donor insemination with frozen semen.

Although it is now accepted that cryopreserved semen must, on ethical and medicolegal grounds, be used for donor insemination many clinicians still believe that it has an unacceptably reduced fecundability rate as compared with fresh semen. We studied the outcome of 81 recipients who started therapeutic donor insemination (TDI) treatment during 1986 in a program that used exclusively cryopreserved semen; 55 had never undergone TDI and were receiving the first series (six cycles), 6 were receiving the second series (also six cycles), and 20 had achieved pregnancy through TDI previously and were starting the treatment again. Insemination with semen stored in 0.5-ml French straws was performed daily during the periovulatory period while the modified Insler score was 10 or greater out of 15. A total of 42 (52%) of the recipients became pregnant within six TDI cycles; 4 (10%) had a spontaneous abortion. An average of 4.8 straws were used per cycle among those who became pregnant and 5.1 per cycle among those who did not. On average 2.6 cycles were required to achieve pregnancy. The overall fecundability rate was 14.6%. We conclude that a TDI program involving exclusively frozen semen can be operated with a success rate comparable to rates achieved with fresh semen if a simple, established cryopreservation method and an uncomplicated clinical management protocol are used.     

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.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

Intrauterine insemination enhances fertility of frozen semen in superovulated ewes

Superovulated ewes were inseminated with fresh or frozen semen in a factorial experiment which compared two techniques of artificial insemination; i.e. conventional cervical deposition and intrauterine deposition at laparoscopy. Similar fertilization rates resulted from insemination with fresh semen at cervical (81% of ova from 11/11 ewes) and intrauterine (83% of ova from 10/12 ewes) sites. These results approached those observed in a naturally-mated group (95% of ova from 5/5 ewes). In ewes inseminated with frozen semen, fertilization rate was markedly reduced (P less than 0.05) after cervical insemination (11% of ova from 3/11 ewes) and partly restored (P less than 0.05) after intrauterine insemination (50% of ova from 8/11 ewes).     

Management and fertility of mares bred with frozen semen.

Semen quality, mare status and mare management during estrus will have the greatest impact on pregnancy rates when breeding mares with frozen semen. If semen quality is not optimal, mare selection and reproductive management are crucial in determining the outcome. In addition to mare selection, client communication is a key factor in a frozen semen program. Old maiden mares and problem mares should be monitored for normal cyclicity and all, except young maidens, should have at least a uterine culture and cytology performed. Mares with positive bacterial cultures and cytologies should be treated at least three consecutive days when in estrus with the proper antibiotic. With frozen semen, timing the ovulation is highly desirable in order to reduce the interval between breeding and ovulation. The use of ovulation inducing agents such as human chorionic gonadotropin (hCG) or the GnRH analogue, deslorelin, are critical components to accurately time the insemination with frozen semen. Most hCG treated mares ovulate 48h post-treatment (12-72h) while most deslorelin (Ovuplant) treated mares ovulate 36-42h post-treatment. However, mares bred more than once during the breeding cycle appear to have a slight but consistent increase in pregnancy rate compared to mares bred only once pre- or post-ovulation. In addition, the "capacitation-like" changes inflicted on the sperm during the process of freezing and thawing appear to be responsible for the shorter longevity of cryopreserved sperm. Therefore, breeding closer to ovulation should increase the fertility for most stallions with frozen semen. Recent evidence would suggest that breeding close to the uterotubal junction increases the sperm numbers in the oviduct increasing the chances of pregnancy. Post-breeding examinations aid in determining ovulation and uterine fluid accumulations so that post-breeding therapies can be instituted if needed. Average pregnancy rates per cycle of mares bred with frozen semen are between 30 and 40% with a wide range between sires. Stallion and mare status are major factors in determining the success of frozen semen inseminations. Pregnancy rates are lower for barren and old maiden mares as well as those mares treated for uterine infections during the same cycle of the insemination. To maximize fertility with frozen semen, a careful selection of the stallions and mares, with proper client communication is critical. Dedication and commitment of mare owner and inseminator will have the most significant impact on the pregnancy rates.     

Effect of insemination dose on pregnancy rate in mares

Different insemination doses have been used for artificial insemination(AI) in horses. Since the insemination dose can affect the pregnancy rate, it is important to ensure that an adequate dose be used regardless of the type of inseminationprotocol used. The aim of this study was to find out if it is possible to decrease the insemination dose from 500 x 10(6) progressively motile spermatozoa to 300 x 10(6) progressively motile spermatozoa and still maintain an acceptable pregnancy rate when using extended fresh semen. Thirteen stallions of known fertility and a well-defined group of 64 mares were used in the study. The mares were randomly assigned to 1 of 2 insemination groups. Examination for pregnancy was performed by ultrasonography per rectum approximately 16 d after the last insemination. When using an insemination dose of 300 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 75%. With an insemination dose of 500 x 10(6) progressively motile spermatozoa the pregnancy rate per cycle was 64%. There was no significant difference in the pregnancy rate between the 2 insemination doses (P = 0.341). We conclude that when using fresh extended semen it is unlikely that an insemination dose of 300 x 10(6) progressively motile spermatozoa would yield a lower pregnancy rate than a dose of 500 x 10(6) progressively motile spermatozoa if stallions with good quality semen are selected.     

Effectiveness of frozen boar semen under practical conditions of artificial insemination

A technique of boar semen deep-freezing and frozen semen use was tested in practice. 338 sows and 43 gilts belonging to small herds with less than 10 females each were inseminated without oestrus detection by a teaser boar. About 58 % of the inseminated females produced 9.3 piglets per litter. But there were differences between parities. The sows had the highest fertility rate, whereas the gilts showed a significantly lower farrowing rate (59.8% vs 41.9%; P < 0.05). The standing reaction of the female to the back pressure test made by the inseminator and the behaviour of the female during insemination had an effect on the farrowing rate. The best result was obtained after a standing reaction and a behaviour score of 1 (64.5% and 9.6 piglets for farrowing rate and litters size respectively). Farrowing rate for inseminators ranged from 44.3% to 62.4% among inseminators. Farrowing rate for females inseminated with frozen semen from Large-White, Landrace, Pietrain boars was not different, but there were significant differences between the boars. Results showed that insemination with deep-frozen boar semen could be used under practical conditions as an additional technique to the use of fresh semen.     

Short-duration insemination with frozen semen increases fertility rate in nulliparous dairy goats

Standard artificial insemination (AI) using a speculum in dairy goats does not result in acceptable fertility rates in nulliparous does. An explanation might be the difficulties to pass the cervical canal in nulliparous females with the insemination gun, increasing the time needed for semen deposition. Nulliparous Alpine dairy goats were used to evaluate whether time interval from insertion to withdrawal of the speculum is a factor influencing pregnancy rates to first AI with frozenthawed semen. Oestrus was synchronized using fluorogestone acetate intravaginal sponges (FGA, 40 mg) for 11 days, associated with 50 mg i.m. of cloprostenol and 250 IU i.m. eCG 48 ± 2 h before sponge removal. In the first experiment (n = 52; 3 herds), the average duration of the AI procedure was 42 ± 10 s, with a median of 39 s. AI performed in less than 39 s resulted in higher pregnancy rates (75%, n = 28) than AI lasting for more than 39 s (46%, n = 24). In the second experiment, does (n = 325; 5 herds) were randomly assigned into two treatment groups according to a short (20 s) or long (60 s) AI procedure. We showed that the duration of AI affected fertility after a first insemination, and that pregnancy rate was significantly improved using a short-duration AI (61.2%; n = 169) compared with a long-duration AI (44.2%; n = 156). We have previously shown in the ewe that genital stimulation during AI enhanced uterine motility. Other authors reported a negative correlation between increased uterine motility at the time of AI and fertility rates in small ruminants. The results of this study suggest that rapid semen deposition may limit the reflex activation of uterine contractions provoked by the speculum and the movement of the insemination gun, and thus ameliorates reproductive performance to first AI in nulliparous goats.     

Infection of embryos following insemination of donor mares with equine arteritis virus infective semen

The objective was to evaluate the potential risks associated with embryo transfer from mares bred with equine arteritis virus (EAV) infective semen. Twenty-six mares were embryo donors, whereas 18 unvaccinated and EAV antibody seronegative mares were embryo recipients. Of the 26 donor mares, 15 were unvaccinated and seronegative for antibodies to EAV and 11 were vaccinated for the first time with a commercially available modified live virus vaccine against EVA before breeding and subsequent embryo transfer. All donor mares were bred with EAV-infective semen from a stallion persistently infected with the virus. Twenty-four embryos were recovered 7 d post-ovulation; all were subjected in sequential order to five washes in embryo flush medium, two trypsin treatments, and five additional washes in embryo flush medium (prior to transfer). Twelve and seven embryos (Grades 1 or 2) were transferred from the non-vaccinated and vaccinated donors, respectively, and pregnancy was established in 3 of 12 and 2 of 7. Perhaps trypsin reduced embryo viability and pregnancy rate. The uterine flush fluid of 11 mares (9 of 15 and 2 of 11 from non-vaccinated and vaccinated donor groups, respectively) was positive for EAV by VI (confirmed by real-time RT-PCR); the wash fluid from the embryos of nine of these mares was negative following 10 washes and two trypsin treatments. However, the embryo wash fluid from two mares was still positive for EAV after all 10 washes and the two trypsin treatments, and one embryo was positive for EAV. Two of 18 recipient mares had seroconverted to EAV 28 d after embryo transfer. Virus was not detected in any fetal tissues or fluids harvested after pregnancies were terminated (60 d). In conclusion, we inferred that the washing protocol of 10 washes and two trypsin treatments did not eliminate EAV from all embryos; due to limitations in experimental design, this requires confirmation. Furthermore, there may be a risk of EAV transmission associated with in vivo embryo transfer from a donor mare inseminated with EAV infective semen.     

The effect of semen extender,seminal plasma and raw semen on uterine and ovarian blood flow in mares

Transrectal color Doppler sonography was used to evaluate the effect of intrauterine infusion of skim milk semen extender, seminal plasma and raw semen on the endometrium and blood flow in the uterine and ovarian arteries in mares. Six Trotter mares (mean age: 12 years) were examined during estrus in three cycles. Each mare received an intrauterine infusion of 20 ml of skim milk semen extender, seminal plasma or raw semen during estrus in one of three cycles. Blood flow measurements in both uterine and ovarian arteries and the determination of intrauterine fluid via sonography were performed before each infusion and 1, 3, 6, 12, and 24 h after infusion. Forty-eight hours later, the intrauterine infusion and measurements were repeated using the same time intervals. Changes in blood flow were detected using transrectal color Doppler sonography and were evaluated using the mean time-averaged maximum velocity (TAMV) of the blood flow. Cytological and bacteriological examination of uterine swabs performed 48 h after the second infusion revealed less inflammation and bacterial growth in mares infused with skim milk semen extender than in those infused with seminal plasma or raw semen. There was an increase in intrauterine fluid as early as 1 h after infusion of any of the substances. The infusion of skim milk semen extender had no effect on uterine blood flow. Within 1 h after infusion of seminal plasma or raw semen, there was an increase in the TAMV values of both uterine arteries (P<0.05). In contrast, ovarian blood flow increased only in the artery ipsilateral to the preovulatory follicle and only after the infusion of raw semen (P<0.05). In conclusion, the changes in uterine perfusion observed after intrauterine infusion may be associated with endometrial inflammation and vasodilatory components in the seminal plasma, whereas the changes seen in ovarian blood flow are possibly attributable to the interaction between sperm and oviduct.