The validation and application of an ovine fertility model using standardised in vitro thresholds to predict the likelihood of pregnancy.

Uterine tone influences fertility of Merino ewes following laparoscopic artificial insemination

To improve the fertility of cervical artificial insemination (AI) in sheep, we investigated isoxsuprine HCl usage on the cervical passage during cervical AI. We also compared cervical and laparoscopic AI fertility results of using chilled semen at different durations. Semen was collected from rams and diluted as 20 × 106 or 400 × 106 spermatozoa/straw for laparoscopic and cervical AI, respectively, and chilled to 4°C within 2 h. Sheep were inseminated with chilled semen for 8 or 24 h via the laparoscopic or cervical AI method. Moreover, some of the cervical inseminated sheep were injected intramuscularly with 0.5 mg/kg of isoxsuprine HCl 15 min before AI. As a result, the use of isoxsuprine HCl did not affect cervical transit and fertility. In addition, fertility was affected by the storage duration of the semen; laparoscopic AI was more successful than cervical AI in terms of fertility; if cervical AI is performed, the duration between semen collection and AI should be less than 8 h after chilling the semen at 4°C, and if laparoscopic AI is performed, the time between semen collection and insemination can be up to 24 h after chilling the semen at 4°C. Longer storage periods should be studied.

Artificial insemination (AI) is a valuable tool for infusing genetic variation into stagnant populations, especially using frozen-thawed spermatozoa, and for propagating physically or behaviorally incompatible individuals. Within zoological institutions, AI has received growing interest for genetic management of endangered felids, provided that AI success can be optimized for applied usage. Traditionally, laparoscopic AI in cats involves treatment with equine chorionic gonadotropin (eCG) followed 80-85h later by human CG (hCG) to induce follicular development and ovulation, with subsequent bilateral sperm deposition into the uterine lumen. However, hCG, a large glycoprotein, may remain in circulation for several days post-injection, generating undesirable secondary ovulations. Uterine AI also requires relatively high numbers of spermatozoa to achieve sperm transport through the uterotubal junction and fertilization within the oviduct. Furthermore, sperm recovery from male cats frequently is poor, limiting the number of spermatozoa available for AI. Alterations in the AI protocol, using short-acting porcine luteinizing hormone (pLH) as the ovulatory signal and oviductal AI for sperm deposition, could improve fertilization success while requiring fewer spermatozoa. Our objectives in this study were to assess pregnancy and fertilization success in cats treated with one of two gonadotropin regimens (eCG/hCG vs. eCG/pLH) and inseminated laparoscopically at two sperm deposition sites, the uterus (UT) and the contralateral oviduct (OV). Sixteen female domestic cats were randomly assigned to either eCG (100 IU)/hCG (75 IU) or eCG/pLH (1000 IU) treatment groups. All 16 females ovulated following gonadotropin treatment and were inseminated with low sperm numbers (1x106 motile sperm/site; 5 µl volume) in one uterine horn and one contralateral oviduct using freshly collected semen from a different male for each site. Semen samples were obtained from two males of proven fertility via artificial vagina. Pregnant females were spayed at 20-21 days post-AI and recovered fetuses assessed for paternity using genetic analysis. Comparing gonadotropin regimens, similar numbers of females became pregnant following eCG/hCG (75%, 6/8) versus eCG/pLH (63%, 5/8). The number of corpora lutea (CL) at AI was similar between regimens, but hCG treatment increased the number of CLs at day 20 post-AI. Although hCG and pLH treatments produced similar numbers of normal fetuses, implantation abnormalities (e.g. empty gestational sacs, malformed placentae) were observed in the hCG, but not pLH, group. Overall, 11 (of 16; 69%) females became pregnant (5 cats from OV AI only, 2 from UT AI only, and 4 from both sites). In comparing insemination sites, more fetuses resulted from OV AI (36/49; 73%) than UT AI (13/49; 27%). To assess capacity for term pregnancies, three additional females were treated with eCG/pLH and inseminated in one oviduct and the contralateral uterine horn. All three females became pregnant (2-3 fetuses each) and healthy kittens were produced. In summary, laparoscopic oviductal AI with low sperm numbers in eCG/pLH-treated females resulted in high pregnancy and fertilization percentages in domestic cats. These findings suggest that this technique may have value for propagating endangered nondomestic cat species. Our recent success in using oviductal AI in eCG/pLH-treated ocelots (Leopardus pardalis) to produce a healthy ocelot kitten supports this cross-species applicability. (platform)

Laparoscopic insemination with frozen-thawed semen is currently used for planned matings in the Sarda breeding programme. In order to find a fast and less intrusive artificial insemination (AI) method that could replace laparoscopic insemination, a field comparison of laparoscopic and transcervical techniques was carried out on 200 mature Sarda ewes. After AI, ewes were assigned to teaser and fertile rams for 2 months. Return rates and cumulative (AI + natural mating) lambing rates were recorded over three subsequent 23-day periods. Lambing rates to AI were significantly different (P < 0·01), and were 62% and 7% respectively for laparoscopic and transcervical AI. Cumulative lambing rates after two further 23-day periods of natural mating were no longer significantly different (P > 0·05) and reached 82% and 74% respectively. Ewes with body condition scores at AI higher than 2·75 showed better overall reproductive performance, but not higher pregnancy rate to AI. Plasma cortisol concentrations, sampled twice, before and after AI, were higher (P < 0·01) in the last sample, suggesting a stress response to insemination. Cortisol levels after AI were lower (P < 0·01) for ewes submitted to transcervical rather than laparoscopic insemination (P < 0·01). However, cortisol levels after AI were no greater than those recorded when ewes were restrained in a milking yoke different from that usually employed. Laparoscopic AI was confirmed as the most suitable technique for insemination offrozen semen in the Sarda breeding scheme.

The present study was conducted in crossbred ewes to compare the efficiency of different artificial insemination techniques with natural tupping following fixed time artificial insemination using fresh semen. Crossbred ewes (n=29) were randomly divided into three groups with 10 animals in natural tupping (NT) and laparoscopic artificial insemination (LAI) groups and nine animals in cervical artificial insemination (CAI) group. Ewes in all the treatment groups were subjected to the same estrus induction protocol: insertion of intravaginal progesterone sponges for 10 days followed by an intramuscular injection of 500 IU eCG at the time of sponge withdrawal. Immediately after removal of sponges, animals of NT group were kept with proven breeding rams up to 72 h. Fixed time cervical or laparoscopic insemination was done at 48 h after sponge removal. The insemination was repeated 12 h later in CAI group. The pregnancy and lambing rates were significantly higher in NT group (90%, 90%), than CAI group (55.5%, 55.5%) and LAI group (20%, 10%). The prolificacy rate was significantly higher in CAI group (180%) than NT group (111.1%) and LAI group (100%). The serum progesterone concentration was higher in pregnant ewes on day 10, day 17 and day 35 than non-pregnant ewes. The fixed time cervical insemination following intravaginal progesterone sponges for 10 day+eCG protocol resulted in better pregnancy rate and prolificacy rate in crossbred ewes during breeding season.

The causes of variation in the success of laparoscopic artificial insemination (AI) in sheep are not well understood. As such, this study incorporated the contributions of multiple male and female factors relevant to the success of AI into a comprehensive prediction model for pregnancy success. Data from Merino ewes (N = 30 254) including age, uterine tone (1; pale/flaccid-5; turgid/pink), intra-abdominal fat (1; little to no fat present-5; high fat), time of insemination and sire used, were recorded during AI. A subset of semen per sire (N = 388) was thawed and assessed for volume, subjective motility, sperm concentration, and morphology. Sperm motility (CASA), viability and acrosome integrity (FITC-PNA/PI), membrane fluidity (M540/Yo-Pro), mitochondrial superoxide production (Mitosox Red/Sytox Green), lipid peroxidation (Bodipy C11), level of intracellular reactive oxygen species (H2DCFDA) and DNA fragmentation (Acridine Orange) were also assessed 0, 3 and 6 h post-thaw. Logistic binomial regression revealed sperm concentration (P < 0.001), CASA parameters at 0 h (PCA3; P = 0.03), viable acrosome intact sperm at 6 h (P = 0.02), abnormal morphology (P < 0.001), uterine tone (P < 0.001) and intra-abdominal fat (P = 0.03) of ewes influenced likelihood of pregnancy. Results generated will help standardise the pre-screening and selection of semen and ewes prior to artificial breeding programs, reducing variation in the success of sheep AI.

The present study aimed to investigate the fertility of ewes artificially inseminated with three different methods using a synthetic semen extender, AndroMed. The three methods of artificial insemination (AI) were cervical AI with fresh-diluted or frozen-diluted semen at observed estrus, and an intrauterine AI with frozen-thawed semen. A total of 80 ewes were treated with a controlled internal drug release (CIDR) containing 0.3 g progesterone per device for 12 days. In Experiment 1 (26 Suffolk ewes), superovulation was induced with 20 mg follicle-stimulating hormone and 250 IU equine chorionic gonadotropin (eCG) two days and one day before CIDR removal, respectively, during the non-breeding season. In Experiment 2 (54 Suffolk and Suffolk crossbred ewes), an intramuscular injection of 500 IU eCG was administered one day before CIDR removal to synchronize estrus and ovulation during the breeding season. In Experiment 1, fresh-diluted or frozen-thawed semen was deposited into the cervical orifice after estrus detection, and an intrauterine AI with frozen-thawed semen was performed by laparoscopy at a fixed-time basis without estrus detection. Embryos were recovered by uterine flushing 6 days after AI, and the rates of recovered, fertilized (cleaved) ova and embryos at the morula or blastocyst stage were compared among the three AI methods. In Experiment 2, the pregnancy rates after the three AI methods were compared. In Experiment 1, the rates of recovered ova were not significantly different among the three AI methods (52.5-56.7%). The rate of fertilized ova (81.0%) by laparoscopic AI with frozen-thawed semen was significantly higher compared with cervical AI of fresh-diluted (25.5%) or frozen-thawed (3.5%) semen, but the rate of embryos at the morula or blastocyst stage (17.6%) was significantly lower than that of the cervical AI with fresh-diluted semen (69.2%). The rates of ewes yielding fertilized ova were not significantly different among the three groups (44.4, 11.1 and 62.5% for cervical AI with fresh-diluted and frozen-thawed semen and intrauterine AI with frozen-thawed semen). In Experiment 2, the pregnancy rate of ewes intrauterinally inseminated with frozen-thawed semen (72.2%) was significantly higher than those of ewes inseminated cervically with fresh-diluted (5.5%) or frozen-thawed (0.0%) semen. The present results showed that acceptable fertilization and pregnancy rates could be obtained by an intrauterine AI with frozen-thawed semen using a synthetic semen extender (AndroMed), but not sufficient by the cervical AI with either fresh or frozen semen.

Artificial insemination in sheep has vastly increased in popularity as preserved semen is readily available. Laparoscopic artificial insemination (LAI), a surgical procedure, involves deposition of semen directly into the uterus using a laparoscope. We have reviewed LAI, compared benefits to natural breeding and transcervical artificial insemination, described procurement, and provided a cost benefit analysis. LAI is an advanced assisted reproductive technique with several benefits, including higher pregnancy rates requiring fewer sperm per breeding than vaginal or transcervical artificial inseminations. Although LAI involves a higher start-up cost compared to other techniques, it is an economically feasible option. With the continual development of new equipment, techniques, and semen preservation technologies, LAI can enhance herd genetics without investment in expensive genetically superior studs and maintaining them. LAI with an appropriate estrus synchronization protocol results in higher numbers of superior quality lambs per ewe for higher profit margin and higher lambing rates for producers, making LAI a profitable service for a veterinary practice.

Fertility following artificial insemination (AI) programs in sheep is affected by many variables. The aim of this study was to identify extrinsic (year, season), intrinsic (age and breed) and AI procedure related (number of AIs, synchronization protocols, semen preservation method) factors that influence fertility after laparoscopic AI in sheep. Data from 159 laparoscopic AI procedures were categorized according to year (from 2013 to 2017), season (Winter, Spring, Summer and Autumn), breed (Dorper, White Dorper, Texel, Cross-breed), age in months (≤12, 13-24, 25-36, 37-48, ≥49), estrus synchronization protocol (short-term or long-term), cumulative number of AIs (1 to 4) and semen preservation method (frozen or fresh). Frequency analysis using chi-square test was used. Year, age, number of AIs and synchronization protocols did not influence fertility after laparoscopic AI in sheep. Fertility was higher (P&lt;0.05) in sheep inseminated in spring (64.28%) and summer (54.16%) when compared to winter (36.92%) and autumn (32.14%). Dorper breed fertility (58.02%) was higher (P&lt;0.05) than White dorper (25%) and Cross-breed (30.23%). There was a lower (P&lt;0.05) pregnancy rate in sheep inseminated with frozen semen (38.27%) than sheep inseminated with fresh semen (53.84%). In conclusion, season, breed and semen preservation method can influence fertility in sheep after laparoscopic AI.

Ovarian sensitivity to exogenous gonadotropins was assessed in the cheetah (Acinonyx jubatus) and clouded leopard (Neofelis nebulosa) to help optimize artificial insemination (AI). Eighteen female cheetahs were used on 29 occasions and were given i.m. injections of 100, 200, or 400 IU eCG and 100 or 250 IU hCG 80 h later. Twenty-three female clouded leopards were treated i.m. on 27 occasions with 25, 50, 75, 100, 200, or 400 IU eCG followed 80 h later with 75, 140, or 280 IU hCG. Ovaries were examined laparoscopically at 43-48 h after hCG in cheetahs and 39-50 h in clouded leopards. All gonadotropin dosages stimulated ovarian activity in both species, but ovulation success and corpus luteum (CL) morphology varied (p < 0.05) with treatment. For both species, the highest and intermediate eCG dosages resulted in ovulation in a high proportion (72-100%) of females. The lowest eCG dosage, although capable of stimulating follicular development, compromised ovulation and resulted in few (< 26%) postovulatory females. For each species, small CL (2-4-mm diameter) were observed with the highest and lowest eCG dosage, and large CL (5-8-mm diameter) were associated with intermediate eCG dosages. Aged CL (10-12 mm diameter) were observed in 4 of 23 (17.4%) clouded leopards with no prior male exposure, indicating occasional spontaneous ovulation. Nineteen laparoscopic intrauterine AI procedures were performed in eCG/hCG-treated postovulatory cheetahs. Eighteen AI procedures were conducted in eCG/hCG-treated postovulatory clouded leopards. Six of the 13 cheetahs (46%), all in the 200-IU eCG/100-IU hCG group, became pregnant, in contrast to none of the clouded leopards. This study has revealed differences in ovarian activity in two wild felid species as a result of changes in exogenous gonadotropin dosage. Because of this dose-effect response, this comparative approach is necessary to identify a gonadotropin regimen that can mimic "normalcy." Even then, the relatively high AI success in the cheetah compared to the clouded leopard suggests that factors other than ovarian response can dictate the efficiency of assisted reproduction in this taxon.

Background & objectivesThe reproductive efficiency of buffalo bulls is crucial for genetic improvement, herd fertility, and overall productivity. Identifying genetic markers linked to semen traits can thus enhance breeding programs and optimize artificial insemination strategies. The objectives of this study were to estimate variance components and heritability. estimating the breeding values (EBVs), plotting the genetic and phenotypic trends and detection of the molecular genetic associations of FSHR and GH genes using PCR–RFLP with semen traits comprising ejaculate volume (EV), sperms motility (SM), live sperms (LS), abnormal sperms (AS) and sperms concentration (SC) in Egyptian buffalo.MethodsData of 5178 semen ejaculates were collected from 2013 to 2022 from 111 bulls, progeny of 34 sires and 92 dams in two experimental herds. For molecular genetic analysis, a total of 86 buffalo bulls were used to characterize FSHR and GH genes.ResultsThe heritabilities estimates for semen traits were low and moderate, being 0.17, 0.28, 0.27, 0.27 and 0.23 for EV, SM, LS, AS and SC, respectively. Wide ranges of the EBVs were observed, being -0.69 to 1.27 ml for EV, -18.19 to 11.59% for SM, -19.31 to 9.15% for LS, -2.05 to 6.41% for AS and -0.39 to 0.54 × 109 sperms/ml for SC. The averages of EBV throughout different years of semen collection were ranged from -0.26 to 0.43 ml for EV, -9.73 and 3.32% for SM, -9.99 and 3.45% for LS, -0.65 to 0.53% for AS and -0.19 to 0.13 × 109 sperms per ml for SC. The phenotypic trends plotted throughout the experimental period increased for all semen traits except for EV. The GLSM of the semen phenotypic values were ranged from 3.09 to 3.86 ml for EV, 61.55 and 66.53% for SM, 60.91 and 65.12% for LS, 4.34 to 9.28% for AS and 0.73 to 1.33 × 109 sperms per ml for SC.The differences in generalized least square means among GG, GC and CC genotypes of FSHR gene for semen traits were significantly in favor of GG genotype relative to GC and CC genotypes (P < 0.01). Two genotypes of TC and CC were detected for GH gene and the molecular genetic associations were significantly in favor of CC genotype relative to TC genotype (P < 0.01).ConclusionEnhancing management and feeding practices, the implementation and widespread use of artificial insemination as well as employing precise estimations of predicted breeding values in genetic improvement programs, should effectively enhance the semen traits of Egyptian buffalo bulls. FSHR and GH genes could be used as potential candidate genes for marker-assisted selection to improve semen traits in buffalo bulls.

Contribution of semen trait selection, artificial insemination technique, and semen dose to the profitability of pig production systems: A simulation study

Context The introduction of bio-sensors for monitoring real-time changes in physiological variables has helped understand how external factors affect an animal’s resiliency to stressors. Aims To quantify changes in temperature, heart rate, and locomotor activity in ewes during hormonal treatments for artificial insemination (AI) for up to 15 days after insemination. Method Twelve ewes received a surgically implanted subcutaneous bio-logger to record data every 5 min. One week later, ewes received an intravaginal sponge for 12 days and AI was performed 54 ± 1 h after sponge withdrawal. The data were divided into the following four periods: ‘sponge in’ (Days -14 to -2), ‘day before AI’ (Day -1), ‘day AI’ (Day 0), and ‘post-AI’ (Days 1–5, Days 6–10, and Days 11–15). Key results Ewes presented significantly (P &amp;lt; 0.001) higher mean temperature and activity, and a lower heart rate when the sponges were in place than they did in the days following AI. Mean body temperature in the ‘sponge in’ period and the ‘day before AI’, but not in ‘post-AI period’, differed significantly (P &amp;lt; 0.001) between pregnant and non-pregnant ewes. Non-pregnant ewes had a significantly (P &amp;lt; 0.001) higher heart rate than did pregnant ewes when sponges were in and in the ‘post-AI’ period. Non-pregnant ewes were significantly (P &amp;lt; 0.001) less active than were pregnant ewes in the ‘sponge in’ period and on Days 1–5 after AI; however, the former were significantly (P &amp;lt; 0.001) more active than were pregnant ewes on Days 11–15 after AI. Conclusions The subcutaneous bio-logger system documented 24-h variations in body temperature, heart rate, and locomotor activity before and after AI in ewes that had received an estrus-synchronising hormonal treatment. Pregnancy status affected those variables and their circadian fluctuations at the time of the hormonal treatment and in the ‘post-AI’ period. Implications Any device designed for use in the study of Precision Livestock Farming that allows a simple, non-invasive measurement of these variables might provide the basis for the development of a system that could identify females that are in an optimal state for insemination, and provide an early pregnancy prediction system.

The effects of gonadotropin treatment and laparoscopic artificial insemination (AI) on embryo quality, serum progesterone and estradiol concentrations, and luteal progesterone content were examined in the domestic cat. These data were compared to similar historical data reported for naturally estrual, mated queens. All queens in this study (n = 32) were treated with eCG followed by 1) natural breeding (eCG-NB), 2) NB and hCG (eCG-NB-hCG), 3) NB and a sham AI procedure (eCG-NB-sham AI), or 4) hCG and actual AI (eCG-hCG-AI). Queens ovulating in response to treatment were ovariohysterectomized, and oviducts and uteri were flushed to collect embryos. Ovarian structures were recorded, corpora lutea (CL) were excised and evaluated for progesterone content, and serum was analyzed for estradiol-17beta and progesterone. Follicle and CL numbers ranged from 0 to 28 and 2 to 42 per cat, respectively, and treatment means did not differ (p > or = 0.05) among groups. Embryos were recovered from oviducts and uterine horns in all treatment groups, and recovery ranged from 60-96%. Mean embryo number per queen ranged from 8.2 +/- 2.6 to 23.2 +/- 3.8 and did not differ (p > or = 0.05) among groups. However, the proportions of unfertilized oocytes were greater (p < 0.05) for groups treated with hCG and/or artificially inseminated, and the proportion of blastocysts produced (31 of 107, 29.0%) was lower (p < 0.05) in the eCG-hCG-AI group than for any other treatment (range, 59 of 116 [50.9%] to 67 of 116 [57.8%]). Not all queens in each group produced good-quality embryos (eCG-NB, 5 of 5; eCG-NB-hCG, 5 of 8; eCG-NB-sham AI, 2 of 5; and eCG-hCG-AI, 3 of 6). Serum progesterone and estradiol-17beta, and total luteal progesterone per ovary did not differ (p > or = 0.05) among treatments. Compared to historical controls (naturally estrual, mated queens), eCG-NB queens produced > 4 times as many good-quality embryos and blastocysts. Similarly, eCG-hCG-AI-treated queens produced > 4 times the number of oocytes and embryos, although a high proportion of these were poor quality and did not develop to blastocysts. Together, these results indicate that queens treated with eCG are capable of consistently producing many good-quality embryos, at least half of which develop to blastocysts in culture. These data support the use of eCG in felids and suggest that other factors are responsible for reduced pregnancy success and small litter sizes following assisted reproduction.

Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen