Homologous prostatic fluid added to frozen-thawed dog spermatozoa prior to intravaginal insemination of bitches resulted in better fertility than albumin-free TALP

The aim of this study was to determine whether canine prostatic fluid has intrinsic effects resulting in higher fertility than albumin-free Tyrode's albumin lactate pyruvate (afTALP) when added to thawed semen prior to intravaginal insemination. Twenty-four German shepherd bitches were inseminated intravaginally with frozen-thawed spermatozoa to which either homologous prostatic fluid (Group P; 12 bitches) or afTALP (Group T; 12 bitches) was added to give a final insemination volume of 7mL. Each bitch was inseminated daily starting when the vaginal folds first became angular and continuing until the day before a diestrus vaginal smear was first seen. Bitches were spayed about 3 weeks after the onset of diestrus and the number of corpora lutea and the number of conceptuses counted. Group P and Group T bitches were, respectively, inseminated 5.3+/-1.0 and 5.8+/-2.1 times with 48.9+/-8.6 and 50.4+/-8.3 million progressively motile spermatozoa per insemination. Eight Group P bitches and 10 Group T bitches conceived with totals of 76 and 45 conceptuses and 126 and 117 corpora lutea, respectively. Odds of conception were taken as the number of conceptuses divided by (the number of corpora lutea minus the number of conceptuses). After adjustment for the number of progressively motile spermatozoa per day and the random effect of bitch, the addition of prostatic fluid resulted in an increased odds of conception compared to afTALP. This effect decreased as the number of progressively motile spermatozoa per day increased.     

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.     

Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability

This study compared the field fertility outcomes in frozen–thawed (FT) sperm from boar ejaculates with different freezability (good, GFE/poor, PFE) while testing the reliability of the post-cervical artificial insemination (post-CAI) in FT sperm. The assay was conducted over eight months with 86 weaned sows being inseminated by post-CAI. Every ejaculate in a total of 26 from 15 Piétrain boars was divided into a refrigerated semen portion (FS; control treatment) and a cryopreserved portion (FT sperm), and the ejaculates were in turn classified as GFE or PFE in function of the sperm progressive motility and viability at 240 min post-thaw. As result, one of four possible treatments was randomly given to each sow: FS-GFE, FS-PFE, FT-GFE and FT-PFE. The number of pregnant and farrowing sows in FT-GFE did not significantly differ from those of FS control treatments. Contrarily, the probabilities of pregnancy were two times lower after inseminations with FT-PFE (P < 0.05) compared to FT-GFE, which indicates that ejaculates with high post-thaw sperm progressive motility and viability are more likely to result in pregnancies than those with poor in vitro sperm function. There were no differences in litter size or the risk of backflow among treatments. Further trials are required to determine the optimal volume and concentration of FT sperm in post-CAI to obtain a more reliable method for farmers interested in cryopreserved sperm.     

Artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach

From 1994 to 2003, a total of 526 bitches of 99 different breeds were artificially inseminated in 685 estrus cycles with domestic (n = 353) or imported (n = 332) frozen-thawed semen from 368 males. The overall whelping rate was 73.1% and mean (+/- S.E.M.) litter size 5.7 +/- 0.1 pups. The whelping rate was higher after intrauterine insemination (75.0%; n = 665) than after intravaginal insemination (10.0%, n = 20; P < 0.05). Insemination at the optimal time resulted in a higher whelping rate (78%, n = 559; P < 0.01) and larger litter size (5.8 +/- 0.2; P < 0.05) than inseminations performed late or too late (55.7% and 4.5 +/- 0.5, n = 61). Two inseminations (n = 384) yielded a higher whelping rate (P < 0.05) and mean litter size (P < 0.01) than one insemination (n = 241), 78.1% and 6.0 +/- 0.2 and 70.5% and 5.1 +/- 0.2, respectively. For inseminations performed at the optimal time, however, the whelping rate was not significantly different for bitches inseminated twice (79.3%, n = 358) versus once (76.8%, n = 168), but the litter size was larger (6.0 +/- 0.2 and 5.3 +/- 0.3). Semen classified as of poor quality (progressive motility < 50% or percentage abnormal sperm > 20%) resulted in a lower whelping rate (P < 0.01) than semen classified as of good quality (progressive motility > or = 50% and percentage abnormal sperm < or = 20%), 61 and 77%, respectively. Small breeds (n = 50) had a smaller litter size (3.9 +/- 0.3; P < 0.01) than larger breeds (medium [5.7 +/- 0.3, n = 94], large [5.9 +/- 0.2, n = 295] or giant breeds [6.1 +/- 0.5, n = 62] [P < 0.01]). Bitches older than 6 years had a lower whelping rate (68.2%) than younger ones (77.0%; P < 0.05). The duration of pregnancy was longer (P < 0.01) for bitches with a litter size of < 3 pups (61.7 +/- 0. 4 days, n = 30) than for bitches with larger litters (60.5 +/- 0.1 days, n = 177). These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs. The results with frozen semen inseminations were optimised by inseminating bitches < or = 6 years old 2 and 3 days after ovulation with semen of good quality from males < or = 8 years old.     

Laparoscopic intrauterine insemination in the bitch

A technique for laparoscopic intrauterine insemination in bitches is described. During natural estrus, 5 beagle bitches were inseminated and S others were naturally mated (control group) twice at a 48-h interval on Days 3 and S (n = 4) or Days 4 and 6 (n = 6) after the increase in plasma progesterone considered to be indicative of the day of the preovulatory LH peak. All the inseminations were with fresh semen and under general anesthesia. The technique involved the introductions of 1) a Verres needle to insufflate the abdominal cavity by direct punction on the middle line 1 cm over the umbilicus, 2) a laparoscope to visualize the abdominal cavity by a 1 cm puncture on the middle line 1 cm under the umbilicus, 3) a forceps used to manipulate the uterus by a 0.5 cm puncture at 2 to 3 cm lateral to the mammary glands, and 4) an 18-g catheter used to puncture the uterus on the middle line between the 3rd and 5th mammary gland. The uterine body was grasped by the forceps and elevated against the ventral abdominal wall. The 18-g catheter was then inserted through the abdominal wall directly into the uterine lumen, and 1.0 ml of fresh semen containing 250 to 480 x 10(6) spermatozoa/ml was injected. The inseminations resulted in pregnancies in all animals. Litter size was similar in the artificially inseminated and naturally mated bitches (5 +/- 1.8 and 4.8 +/- 1.6 pups per litter, respectively). Bitches in the artificially inseminated group delivered at 65.2 +/- 0.8 d and in the natural mated group at 65.4 +/- 0.5 d after the LH peak. In conclusion, this paper gives the first results of intrauterine laparoscopic insemination in bitches, indicating interesting perspectives for this technique in dog's reproduction.     

Comparison of endoscopic-assisted transcervical and laparotomy insemination with frozen-thawed dog semen: A retrospective clinical study

The objective of this retrospective clinical study was to compare pregnancy rates obtained after the use of endoscopic-assisted transcervical catheterization (EIU) or laparotomy (SIU) for insemination of frozen-thawed dog semen. Healthy bitches from various breeds were inseminated with semen from multiple donors processed by different freezing centers. Data from 118 inseminations (78 EIU and 40 SIU) performed between 2009 and 2011 were analyzed. Insemination timing was based on vaginal cytology, serum progesterone concentrations, and vaginoscopy. A ureterorenoscope and a CH-5 Transcervical insemination catheter were used for EIU; 28 of the bitches in this group were inseminated twice with the second insemination less than 12 hours after the first. The numbers of live morphologically normal sperm (LMNS) were determined to characterize insemination doses. Overall, pregnancy rate was greater (P < 0.05) in the EIU group (65%) than in the SIU group (45%). Pregnancy rates were greater (P ≤ 0.06) when more than 100 × 10⁶ LMNS were inseminated regardless of insemination method; the greatest pregnancy rate was observed in the EIU group when this insemination dose was used (38/49; 78%). There was no significant difference in pregnancy rate whether one (69%) or two inseminations (64%) were performed in the EIU group. Complications in the SIU group included anesthetic-induced bradycardia during surgery, significant postsurgery pain, seroma formation over the abdominal incision, and delayed wound healing. No complications were noted during or after insemination in the EIU group. In conclusion, these results support the use of EIU as a noninvasive alternative to laparotomy for insemination of frozen-thawed dog semen. In addition, use of more than 100 × 10⁶ LMNS is also recommended for insemination.     

Comparison of efficiency between two artificial insemination methods using frozen–thawed semen in domestic cat (Felis catus): Artificial insemination in domestic cats

The aim of this study was to compare the efficiency of the intravaginal (IVAI) vs. intrauterine artificial insemination (IUAI) using frozen–thawed sperm in the domestic cat. Semen was collected from two tom cats using an artificial vagina and samples were assessed for motility (computer-assisted sperm analysis (CASA)), sperm morphology and plasma membrane integrity. After dilution with TRIS/OEP/YOLK (4% of glycerol), sperm samples were loaded into 0.25 mL straws (25 × 10⁶ motile sperm/straw), incubated at 5 °C for 20 min and cryopreserved over liquid nitrogen (LN₂) vapor for 15 min and then immersed in LN₂. For each AI, four straws from the same male were thawed (12 s at 46 °C) and centrifuged at 250 × g for 8 min to pellet the sperm. The supernatant was discarded and sperm pellet resuspended with the remaining liquid, approximately 100 μL, and analyzed as described above. Queens were treated with a single im injection of 100 IU eCG to induce ovarian follicular development. Final oocyte maturation and ovulation was induced with 100 IU hCG given im at 82–84 h after eCG administration. Thirty hours after hCG administration, females were inseminated either intrauterine (n = 8 queens) or intravaginally (n = 8 queens), using thawed sperm from a single male. Although a pronounced decrease in sperm motility, acrosome and plasma membrane integrity was observed in sperm samples from both cats, a pregnancy rate of 75% was achieved when using the intrauterine AI method compared with 0% pregnancy when inseminated intravaginally.     

Semen preservation and artificial insemination in domesticated South American camelids

Semen preservation and artificial insemination in South American camelids are reviewed giving emphasis to work done in Peru and by the authors. Reports on semen evaluation and the preservation process indicate that semen of alpacas and llamas can be manipulated by making it liquid first. Collagenase appears to be the best enzyme to eliminate viscosity. Tris buffer solution maintains a higher motility than egg-yolk citrate, phosphate buffered saline (PBS), Triladyl, and Merck-I extenders. Cooling of semen took 1 h after collected, and equilibrated with 7% glycerol presented a better motility and spermatozoa survival at 1, 7, 15 and 30 days after being slowly frozen in 0.25 mL plastic straws. Trials of artificial insemination with freshly diluted semen and frozen–thawed semen are encouraging and needs to be tested extensively under field conditions. Recently, fertility rates varied from 3 to 67%. Semen preservation and most important, artificial insemination appear to be a reality, and could be used to improve the genetic quality of alpacas and llamas.     

Artificial insemination in domestic cats (Felis catus)

Artificial insemination (AI) in cats represents an important technique for increasing the contribution of genetically valuable individuals in specific populations, whether they be highly pedigreed purebred cats, medically important laboratory cats or endangered non-domestic cats. Semen is collected using electrical stimulation, with an artificial vagina or from intact or excised cauda epididymis. Sperm samples can be used for AI immediately after collection, after temporary storage above 0 degrees C or after cryopreservation. There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report on tubal insemination with fresh semen. In studies using fresh semen, it was reported that conception rates of 50% or higher were obtained by intravaginal insemination with 10-50x10(6) spermatozoa, while, in another report, the conception rate was 78% after AI with 80x10(6) spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2x10(6) and 8x10(6) spermatozoa were reported to be 50 and 80%, respectively. With tubal insemination, the conception rate was 43% when 4x10(6) spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that of cats inseminated directly into the uterus. When frozen semen was used for intravaginal insemination the conception rate was rather low, but intrauterine insemination with 50x10(6) frozen/thawed spermatozoa resulted in a conception rate of 57%. Furthermore, in one report, conception was obtained by intrauterine insemination of frozen epididymal spermatozoa. Overall, there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within and among laboratories depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly, future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a practical technique for enhancing the production of desirable genotypes, both for laboratory and conservation purposes.     

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.     

Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality,fertilization, and hatching

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2 × 2 × 2 × 5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8–31.5%) of fertile, non-viable embryos (Day 1–6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10–13 weeks and 9–10 weeks, respectively. The duration of hatchability was 4–6 weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5 ± 2.4%), followed by the E line (5.3 ± 1.3%), F line (3.7 ± 2.0%) and RBC2 line (2.6 ± 0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.     

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank’s balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen–thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen–thawed-graft groups (p < 0.05). Fresh grafts and frozen–thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p > 0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p < 0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p < 0.05). The typical damage observed in the frozen–thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.     

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 preparing and loading multiple insemination guns on conception rate in two large commercial dairy herds

In large herds, inseminators frequently thaw multiple straws of semen and prepare several insemination guns at once. The aim of this study was to measure the effect of breeding order, the order that thawed straws are used, on conception rate in commercial dairy herds. A single professional inseminator, utilizing semen from five suppliers, performed 2629 inseminations over 30 months. Up to four straws were thawed at one time with the restriction that straws were used within 20 min of thawing. Straws were thawed per supplier's recommendations, with 66.4% of the straws pocket thawed and the remainder warm-water thawed. Conception was determined by a pregnancy check at 42 days. Data were modeled by multiple logistic regression analysis, which included herd, breeding order, lactation number, times bred, month bred and year bred. Breeding order had no significant effect on the probability of conception: Cows 1-4 achieved adjusted conception rates of 0.36, 0.41, 0.37, and 0.39, respectively. Odds ratios (and 95% confidence intervals) were 1.00 (1.00-1.00), 1.22 (0.99-1.49), 1.04 (0.82-1.32), and 1.12 (0.86-1.45), respectively. Associated laboratory studies, which evaluated the effect of post-thaw holding time on percentage of sperm with progressive motility, found mean values at 20 min holding time to be largely unchanged from mean values at 5 min. Thawing temperatures were 6, and 35 degrees C and holding temperatures were 6, 22, and 35 degrees C. The length of the trial and the wide use of semen from multiple suppliers with differing thawing methods suggests that under similar conditions, a careful and experienced inseminator can thaw multiple straws of semen and prepare insemination guns sufficient to breed up to four cows within 20 min, without an adverse effect on conception.     

Esterified glucomannan improves aflatoxin-induced damage of sperm parameters during liquid storage of ram semen at 5 °C

The aim of the present work was to study the effects of aflatoxin (AF) on sperm parameters in rams, and to determine the protective efficiency of esterified glucomannan (EG) co-administered with AF up to 96 h of the liquid storage of ram semen at 5 °C. Thirty-two Merino rams (12–14 months old) were used. The animals were examined for their general health status. To ensure their adaptation to the environment and the new feeding regimen, a 15-day acclimatization programme was applied to the animals, prior to the start of the study. Experimental feeding was continued for ninety-two days. The experimental design consisted of four dietary treatments. The control group (C) was fed with commercial feed. The AF group was fed with commercial feed plus 250 μg/day of total AF. The EG group received commercial feed plus 2 g/day of EG. The AF + EG group was given commercial feed plus 250 μg/day of total AF and 2 g/day of EG. In the study, ejaculates were obtained from rams twice a week for 12 weeks, using an electro-ejaculator. After collected, the ejaculates were diluted with a skimmed milk extender, and stored at 5 °C. Sperm motility and rates of abnormal and nonviable spermatozoa were determined for the different treatment groups at 5 °C at 0, 24, 48, 72 and 96 h of liquid storage.     

Alterations of the pituitary-ovarian axis in dogs with a functional granulosa cell tumor

Information on the pituitary-ovarian axis in dogs with a granulosa cell tumor (GCT) is lacking. Therefore, we investigated the plasma concentrations of luteinizing hormone (LH) and estradiol before and after gonadotropin-releasing hormone (GnRH) administration in seven bitches with a functional GCT (GCT-total), of which three were intact (GCT-intact) and four had remnant ovarian tissue (GCT-ROT). The results of the GnRH stimulation test were compared with those in six anestrous and six ovariectomized bitches. The most noteworthy results were as follows. The basal plasma LH concentrations of the GCT-ROT bitches were higher (P < 0.05) than those of the anestrous bitches. The increment in the plasma LH concentration after GnRH administration in the GCT-total bitches was lower (P < 0.001) than the increments in both the anestrous and ovariectomized bitches. The basal plasma estradiol concentrations in the GCT-total bitches were higher (P < 0.001) than those in the anestrous and ovariectomized bitches. In conclusion, the pituitary-ovarian axis is affected in bitches with a functional GCT and is characterized by relatively high plasma LH concentrations in GCT-ROT bitches and a subnormal LH response to GnRH stimulation in all GCT bitches compared with those in anestrous and ovariectomized bitches. The relatively high proportion of dogs with remnant ovarian tissue among the GCT bitches suggests a pathogenetic role for elevated gonadotropin secretion in the pathogenesis of GCT.     

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen–thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5 ± 5.4 × 10⁶ sperm (range, 6.8–22 × 10⁶) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 × 10⁶ sperm, with 70 ± 5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17β and progesterone were determined in most queens on the day of AI and again 30–40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P = 0.58); overall 33% (5/15) of the queens became pregnant. For frozen–thawed semen, AI was consistently done 28 h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P = 0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen–thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes

The effect of carazolol on the ease of penetrating the cervix during artificial insemination, lambing rate and litter size was studied using 1.5–4.0-year old Kivircik ewes in an incomplete 3 × 2 × 2 experimental design. All of the ewes in this study were synchronized for oestrus by insertion of a progesterone impregnated vaginal sponge for 12 days and administration of 400 IU PMSG at sponge withdrawal. Three methods of service were compared: natural service, artificial insemination (AI) with fresh semen, or AI with frozen semen. Two times of insemination (fixed time AI versus AI at observed oestrus) were compared on the fresh and frozen AI treatments. The absence (control) or use of carazolol (carazolol; 0.5 mg/ewe i.m. 30 min before mating) was the third factor in the design and penetration of the cervix by the insemination pipette was assessed as shallow (<10 mm), middle (10–20 mm) or deep (>20 mm). Natural service ewes were only mated at observed oestrus. Consequently, the factorial design was incomplete and there were a total of 10 treatments each represented by 30 ewes. Natural service resulted in a significantly (P < 0.05) higher lambing rate and litter size (86%; 2.0 ± 0.05 lambs/ewe) than AI using fresh (65%; 1.6 ± 0.1 lambs/ewe) or frozen (40%; 1.4 ± 0.14 lambs/ewe) semen. For AI animals the lambing rate and litter size were not significantly different when service was at a fixed time (50%; 1.5 ± 0.12 lambs/ewe) or at observed oestrus (56%; 1.5 ± 0.12 lambs/ewe). Carazolol did not permit complete cervical penetration in any ewe. Deep penetration of the cervix at AI was achieved in 33% of untreated (control) and 48% of carazolol treated ewes (P < 0.05). However, the proportion of ewes in which penetration of the cervix and semen deposition was greater than shallow was similar for control (82%) and carazolol (85%), and lambing rate and litter size were similar for both treatments. Over the three service methods, the lambing rate was 56% for control and 63% for carazolol (NS) and litter size was similar for both treatments. It was concluded that the carazolol treatment used prior to natural mating or AI in this experiment did not improve lambing rate or litter size in Kivircik ewes.     

Effect of milk- and TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with liquid semen

We studied the influence of two different extenders, a milk-based versus a TRIS-based extender, using a split-sample technique, on fertility after single and double vaginal inseminations in natural estrous in Norwegian Crossbred ewes. Semen from 21 Norwegian Crossbred rams, all aged approximately 0.5 years, was used for AI of totally 561 Norwegian Crossbred ewes housed at 37 different farms. The farmers performed the inseminations themselves. The ewes were allocated to four parallel groups based on the two extenders and single or double inseminations (2 x 2). The farmers were recommended to inseminate the ewes between 12 and 24 h after detection of natural standing estrous. Vaginal insemination with cooled liquid semen diluted in the milk-based extender resulted in a statistically significant (P<0.01) better fertility of about 10% units both as 25-day NR (non return rate)-and lambing rates, compared with semen diluted in the TRIS-based extender. Double inseminations gave significantly higher (P=0.03) fertility results for both extenders expressed as 25-day NR results, but was not quite statistically significant when expressed as lambing rates (P=0.06) compared with single insemination. The overall 25-day NR results for the milk-based extender (66.4%) after single inseminations is in accordance with both the national results (67.1%) based on vaginal inseminations of 11,377 ewes, as well as with the results from a previous study in the same region achieving a 25-day NR results of 63.3%. In conclusion, liquid ram semen diluted in a milk-based extender and vaginally inseminated once in natural heat, with a semen dose of 150 x 10(6) spermatozoa, gave acceptable fertility results and is to be recommended as the method of choice in Norway.