Ram seminal plasma improves pregnancy rates in ewes cervically inseminated with ram semen stored at 5 °C for 24 hours

In this study, we compared pregnancy rates obtained using ram semen stored at 5 °C for 24 h, with ram or bull seminal plasma (SP) added to TRIS-egg yolk extender. During the breeding period, 670 adult Corriedale ewes were cervically inseminated with semen (2 × 10⁸ sperm in a volume of 0.2 mL) from eight adult Corriedale rams. Ejaculates, obtained using an artificial vagina, were split into three aliquots and diluted with the following: TRIS-egg yolk based extender (T), T + 30% ram SP (R), or T + 30% bull SP (B). Samples were refrigerated and stored at 5 °C for 24 h until used for AI. Pregnancy was assessed by ultrasonography 35 to 40 d after AI. Pregnancy rate was not affected by ram (P = 0.77) or breeding period (P = 0.43), and there were no interactions between extender and ram (P = 0.94), or extender and breeding period (P = 0.24). However, there was an effect of extender (P = 0.0009) on pregnancy rates; ram SP, but not bull SP, increased pregnancy rates compared with extender without SP (49.7, 38.1, and 31.1%, for R, B, and T respectively). In conclusion, ram SP added to TRIS-egg yolk extender had a beneficial effect on the pregnancy rate of ram sperm stored at 5 °C for 24 h and used for cervical insemination of ewes.     

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

Several procedures have been proposed to assess structural and functional characteristics of cryopreserved ram semen but none so far have yielded consistent relationships with in vivo fertility. The objectives of this study were to evaluate several sperm function tests as potential markers of in vivo ram fertility (determined by pregnancy rate in ewes) using frozen-thawed semen. In experiment 1, frozen-thawed straws (n=3 per ram) of semen from three high and three low fertility rams were assessed using fluorescent microscopy for (1) progressive motility, (2) viability and, (3) acrosomal status. In experiment 2, frozen-thawed straws (n=3 per ram) of semen from 18 rams of known fertility were analysed using either computer-assisted sperm analysis (CASA) for eight motion characteristics or flow cytometric staining for: (1) viability and acrosomal status, (2) plasma membrane status and capacitation-like changes, and (3) live cells following an osmotic resistance test (ORT). In experiment 3, platelet-activating factor (PAF) was isolated from straws (n=2 per ram) of semen using high-pressure liquid chromatography (HPLC) and quantified using HPLC-tandem mass spectrometry for 18 rams. In experiment 1, no association was found between motility, viability (% live) or acrosomal status (% damaged, % intact and % reacted) and in vivo fertility. In experiment 2, no correlation was found between motility (CASA), viability (% live), acrosomal status (% live, % live intact and % reacted), capacitation status (% capacitated, % non-capacitated), plasma membrane stability (% dead) and % live cells following ORT and ram in vivo fertility. In experiment 3, there was no relationship between PAF content in spermatozoa and ram fertility. In conclusion, we were unable to relate the in vivo fertility of rams with in vitro functional tests of their frozen-thawed semen and suggest that the fertility of a given semen sample cannot easily be quantified using available in vitro tests.     

Sources of variation in the reproductive performance of ewes inseminated with frozen-thawed ram semen by laparoscopy

Fertility data from 8 artificial insemination programs, involving more than 5000 ewes and 110 rams in 3 flocks, were analyzed to determine variation due to individual AI program and ram in the reproductive performance of ewes inseminated with frozen-thawed semen by laparoscopy. The semen had been previously frozen by commercial AI centers in either pellets or straws. Both AI program and individual ram affected the proportion of ewes pregnant and the number of fetuses per ewe inseminated, but not the number of fetuses per pregnant ewe. Semen samples from 97 of the rams used were analyzed on a Hamilton Thorn HTM 2000 image analyzer for sperm concentration, percentage of motile and progressively motile spermatozoa, mean progressive velocity, and mean linear index. The correlations between these traits and reproductive performance obtained after insemination were calculated. There was large variation in the quantity and quality of the frozen semen, but only the number of total and motile spermatozoa inseminated per ewe was correlated with fertility (0.25 and 0.26, respectively). Regression analysis showed that none of the traits measured were useful for predicting fertility.     

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.     

Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination

Ewe breed has been shown to have a major effect on pregnancy rates following cervical AI using frozen-thawed semen. The main objective of this study was to examine the differences between purebred Belclare and Suffolk ewes (multiparous) in fertilization rate, number of accessory sperm and stage of embryo development on day 6 after cervical or laparoscopic AI with frozen-thawed semen. In experiment 1, Belclare and Suffolk ewes were synchronized for 12 days and were either cervically inseminated (year 1: n=28 and 31; year 2: n=16 and 15, respectively) or laparoscopically inseminated (year 2: n=13 and 14). In experiment 2, superovulated Belclare (n=4) and Suffolk (n=13) ewes were laparoscopically inseminated. All ewes were slaughtered 6 days after AI; oocytes/embryos were recovered, morphologically graded and stained to assess the number of cells and accessory spermatozoa. Data from both experiments were combined for statistical analysis. The proportion of ewes with fertilized oocytes was significantly higher following laparoscopic AI compared with cervical AI (54% versus 19%). More Belclare than Suffolk ewes yielded fertilized oocyte(s) after cervical AI (34% versus 10%, P<0.02) but there was no difference after laparoscopic AI (62% versus 60%). From the ewes that yielded at least one fertilized oocyte the proportion of Belclare ewes with embryos at the morula/blastocyst stage was significantly greater than for Suffolk ewes (94% versus 59%, P<0.02). A higher proportion of Belclare than Suffolk ewes had evidence of sperm reaching the site of fertilization following cervical AI (39% versus 15%, P<0.02) but there was no difference after laparoscopic AI (62% versus 64%, P>0.8). Amongst the ewes with evidence of sperm at the site of fertilization, laparoscopic AI resulted in a higher number of sperm per oocyte/embryo or per ewe than cervical AI (P<0.01). These results suggested that the difference in pregnancy rate between Suffolk and Belclare ewes following cervical AI was due to: (i) sperm traversing the cervix and uterus in a higher proportion of Belclare than Suffolk ewes, leading to a higher incidence of fertilization and (ii) the lower developmental competence of fertilized oocytes from Suffolk ewes.     

Birth of offspring of pre-determined sex after artificial insemination of frozen-thawed, sex-sorted and re-frozen-thawed ram spermatozoa

The fertility of ram spermatozoa cryopreserved prior to, and following, sex-sorting by flow cytometry was assessed after insemination of mature Merino ewes at a synchronised estrus. Ewes were inseminated with spermatozoa from three rams, split into four treatment groups: 50 x 10(6) motile non-sorted, frozen-thawed (Control50), 15 x 10(6) motile non-sorted, frozen-thawed (Control15), 15 x 10(6) motile sex-sorted, frozen-thawed (SF15) or 15 x 10(6) motile frozen-thawed, sex-sorted, re-frozen-thawed (FSF15) ram spermatozoa. Separation of SF15 and FSF15 treatments into X- and Y-chromosome-bearing populations was achieved using a high-speed sperm sorter. The percentage of ewes lambing after insemination was similar for Control15 (36/74; 48.6%), SF15 (35/76; 46.1%) and FSF15 (26/72; 36.1%) groups (P>0.05). A higher percentage of ewes produced lambs in the Control50 (38/70; 54.3%) than the FSF15 group (P<0.05). Fifty-one of the 55 (92.7%) lambs derived from fresh, sex-sorted frozen-thawed spermatozoa were of the predicted sex, as were 41/43 (95.3%) lambs derived from frozen-thawed, sex-sorted, re-frozen-thawed spermatozoa. This study demonstrated for the first time in any species that frozen-thawed spermatozoa, after sex-sorting and a second cryopreservation step, are capable of producing offspring of the predicted sex following artificial insemination.     

In vitro fertilization as a predictor of fertility from cervical insemination of sheep

The objective of this study was to determine if the quality of frozen-thawed ram semen could be effectively evaluated through in vitro fertilization (IVF) procedures prior to insemination as a means of improving pregnancy rate. In experiment 1, frozen semen from four Belclare rams was assessed using IVF and was used for cervical insemination of ewes (n = 181) in 13 pedigree Belclare flocks. There was a significant association between IVF score (proportion of oocytes cleaved at 48 h post insemination) and non-return rate (P < 0.001). For experiment 2, semen from nine Belclare rams was evaluated by IVF and semen from rams with the highest (n = 3) and lowest (n = 2) IVF scores was used for cervical insemination of ewes (n = 111) under experimental conditions. Differences in pregnancy rates between individual rams did not reach significance. Experiment 3 was designed to determine if differences detected between rams at field level could be accurately identified via IVF evaluation and involved frozen semen from eight Norwegian rams of known field fertility (non-return rates ranged from 45.7 to 73.8%). IVF score did not reflect the differences in field fertility. In the final experiment six of the eight Norwegian rams involved in experiment 3 were selected based on IVF score (three highest and three lowest) and their semen was used for cervical insemination (n = 90 ewes). While significant differences in pregnancy rate were found between individual rams (P < 0.02, range: 12.9-65.8%) they were not associated with IVF score. Ewe breed had a significant effect (P < 0.003) on pregnancy rate in both experiments 2 and 4. In conclusion, there was no evidence from this study that the evaluation of semen quality through IVF provided a useful predictor of pregnancy rate under field conditions. It may be that the IVF procedures as used routinely, which are essentially designed to maximize blastocyst yields rather than for detecting differences in fertilizing ability between batches of sperm, need to be modified.     

Laparoscopic insemination of frozen-thawed semen into one or both uterine horns without regard to ovulation site in synchronized Merino ewes

Synchronized ewes (n = 217) were bred by laparoscopic insemination of frozen-thawed semen from 1 of 3 rams. The ewes were bred by either a double (110 ewes) or single horn (107 ewes) technique without regard to the site of ovulation. There was no difference in the percentage of ewes pregnant to either the single or double horn breeding technique. There was a significant effect of sire, with 1 ram producing a higher pregnancy rate in the ewes and 1 ram producing a significantly lower pregnancy rate when compared to the total pregnancy rate for all the ewes (P < 0.05). Thus, the single horn breeding technique is presented as an alternative technique for use in the commercial breeding of ewes by laparoscopic insemination of frozen-thawed semen.     

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57 h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.     

Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination

We compared the fertility of thawed ram semen, frozen according to different prefreezing semen handling protocols and previously well-defined in vitro, after cervical artificial insemination (AI) during natural estrus in Corriedale sheep. Following primary extension 1 + 1, we adjusted the final sperm concentration before packaging (200 x 10(6)/straw) either by centrifugation, in order to reconcentrate the extended semen (Protocol 1: P1), or without centrifugation, by adjusting the final sperm number by stepwise extension (Protocol 2: P2). We evaluated sperm motility (assessed both subjectively and with a computer-assisted sperm analysis instrument [CASA]), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline [CTC]) in vitro in three pooled straws of frozen-thawed semen. Three hundred Corriedale ewes, having shown spontaneous estrus during the breeding season (i.e., April, in the southern hemisphere) under extensive management conditions in Uruguay, were cervically inseminated with thawed semen from the same freezing operations as studied in vitro. The semen evaluation in vitro yielded higher percentages (P < 0.05) of damaged spermatozoa in the samples where sperm numbers were adjusted by extension before freezing (P2), compared with when adjustment was done by centrifugation (P1). However, due to the higher sperm concentration finally achieved by P2, the calculated total number of viable spermatozoa was almost equal in the two AI doses. We observed no differences in fertility between P1 and P2 for either nonreturn rates (NRRs) 21 (30.8 vs. 29.7%) and 36 (28.5 vs. 27.8%) days after AI or lambing rate (21.9 vs. 21.4%), respectively. Fertility did not differ significantly between the two different procedures of adjusting sperm numbers prior to freezing. This may indicate that the simplified protocol with adjusted extension of the semen, resulting in higher numbers of viable spermatozoa, should be the procedure of choice when freezing ram semen under field conditions. Further studies aimed at improving the modified protocol need to be performed.     

Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronised oestrus

Artificial insemination (AI) in sheep is currently limited by the poor fertility obtained following non-surgical intracervical insemination of frozen-thawed semen. An exception to this general finding is the non-return rate of around 58% reported for large scale on-farm AI in Norway. The objective of the present study was to determine if similar results could be obtained under Irish conditions. Comparisons were made between semen collected, and frozen, from rams in Norway (NOR) and Ireland (IRL). The effects of synchronisation and inseminator were also examined. Parous ewes (n=297) of various breed types were inseminated to a natural (N) or synchronised (S) oestrus with either fresh (from Irish rams) or frozen-thawed (IRL and NOR) semen. Ewes were randomly assigned, within breed, to the following treatment groups: (i) Fresh-N: n=28, (ii) Fresh-S: n=30, (iii) IRL-N: n=62, (iv) IRL-S: n=50, (v) NOR-N: n=68, (vi) NOR-S: n=59. Within each group, ewes were inseminated by an experienced Norwegian or by an Irish inseminator. Pregnancy rate did not differ significantly between ewes inseminated to a natural or synchronised oestrus nor between Norwegian and Irish frozen semen. The proportion of ewes pregnant after insemination with fresh semen was 0.82 and 0.70 (treatments i and ii) compared with 0.40, 0.52, 0.34 and 0.37 (treatments (iii)-(vi)) for frozen semen (P<0.001). Corresponding litter sizes (+/-S.E.), adjusted for ovulation rate, were 2.9+/-0.22, 3.3+/-0.23, 2.2+/-0.21, 1.7+/-0.21, 2.2+/-0.21 and 2.1+/-0.21 (fresh versus frozen; P<0.001). There was an interaction between semen type (fresh or frozen) and oestrus type (N or S) for litter size due to an increased adverse effect of frozen semen on litter size in synchronised ewes (P<0.05). Pregnancy rate was significantly influenced by breed of ewe (P<0.01) and inseminator (P<0.05). These results suggest that ewe breed may be a critical determinant of the potential for the exploitation of cervical insemination of frozen-thawed semen in sheep breeding programmes.     

Fertility of ram semen frozen in Bioexcell and used for cervical artificial insemination

The current use of ingredients of animal origin, such as egg yolk, in semen extenders presents a risk of microbial contamination, and has led to the search for alternatives. Such an extender is commercially available for bull semen (Bioexcell), IMV, L'Aigle, France), and it has previously been tested in vitro for freezing ram semen, with satisfactory results. The aim of the present study was to compare the fertility results of ewes in Uruguay, after cervical insemination with ram semen that was frozen in Bioexcell versus semen frozen in a conventional milk-egg yolk extender (control). Semen from five Corriedale rams was frozen, using a split sample design, in either milk-egg yolk or Bioexcell extender, using a two-step extension method. The sperm parameters assessed after thawing were subjective motility, membrane integrity (SYBR-14/PI), and capacitation status (CTC). Thawed semen was inseminated intracervically once during spontaneous estrus in 970 Corriedale ewes that grazed in natural pastures, under extensive management conditions. Fertility was recorded as nonreturn rates at 21 days (NRR-21) and 36 days (NRR-36) after artificial insemination (AI), as well as pregnancy rate (PR-US, diagnosed ultrasonographically 50 days after AI of the last ewe). Subjective motility was slightly higher in Bioexcell than in the milk extender (47 vs. 46.5%; NS), as was membrane integrity (38 vs. 37.7%; NS) and the percentage of uncapacitated spermatozoa (28.5 vs. 26.3%; NS). There were no statistically significant differences in fertility rates found between Bioexcell and the control extender: NRR-21 (35.9 vs. 33.2%), NRR-36 (34.8 vs. 32.6%), and PR-US (28.4 vs. 27.2%). In conclusion, Bioexcell appears to be an alternative to the conventional milk-egg yolk extender for freezing ram semen, and provides similar fertility results after cervical AI under extensive management conditions. Thus, Bioexcell, containing no additives of animal origin, can offer a safer alternative when frozen semen is used for introducing new genetic material into a flock or a country.     

Cervical versus intrauterine insemination of ewes using fresh or frozen semen diluted with aloe vera gel

This study was conducted at Belen de Escobar, Argentina, in March and April 1987. Experimental work on synchronization of estrus, deep-freeze conservation of ram semen and small fertility trials involving cervical and intrauterine (i.u.) insemination methods was undertaken. A total of 80 Corriedale ewes were used in seven insemination trials. Insemination trials were grouped into two experimental groups for comparison of 1) frozen semen diluted with an experimental extender and a control diluent inseminated cervically or i.u. in synchronized/superovulated ewes and 2) cervical insemination of fresh diluted or frozen semen in ewes inseminated at natural estrus or in ewes that were synchronized/superovulated. An overall ovulation rate of 8.7 +/- 0.5 was obtained by using a superovulatory regimen consisting of 3 mg Norgestomet implants and a total dose of 18 mg follicle stimulating hormone-pituitary (FSH-P). Numbers of ova recovered per ewe following superovulation ranged from 4.3 to 5.4. In experimental Group I, fertilization rates improved when laparoscopic intrauterine AI was used compared with cervical insemination (P<0.05). Fertility rates of i.u. and cervical insemination of frozen semen diluted with the experimental extender showed satisfactory fertilizing capacity. In experimental Group II, a lower number of fertilized ova were recovered from ewes inseminated with frozen semen (P<0.02), irrespective of their estrus manipulation.     

Pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition

In six field trials, doses between 1.0 and 6.0 x 10(6) total sexed, frozen-thawed sperm were inseminated into the uterine body or bilaterally into the uterine horns of heifers and nursing Angus cows 12 or 24h after observed estrus. Except for one comparison in one trial in which uterine body insemination was slightly superior (P<0.05) to uterine horn insemination, there was no significant (P>0.1) difference between sites of semen deposition. Additionally, except for one small study with limited numbers, there was essentially no difference in pregnancy rates in the range between 1.5 and 6 x 10(6) sexed, frozen-thawed sperm per inseminate. Pregnancy rates with smaller doses of sexed sperm averaged about 75% of controls of 20 x 10(6) total frozen-thawed, unsexed sperm. While 1.0 x 10(6) sexed, frozen-thawed sperm per insemination dose resulted in decreased pregnancy rates compared to larger doses, the lesser fertility with sexed sperm could not be compensated by increasing sperm numbers in the range of 1.5-6 x 10(6) sperm per dose. Pregnancy rates with 2 x 10(6) sexed, frozen-thawed sperm per dose were not markedly less than control pregnancy rates with 20 x 10(6) frozen-thawed unsexed sperm/dose in well-managed herds.     

Estrus synchronization and artificial insemination of hair sheep ewes in the tropics

Hair sheep ewes (St. Croix White and Barbados Blackbelly) were used to evaluate 3 methods of estrus synchronization for use with transcervical artificial insemination (TAI). To synchronize estrus, ewes (n = 18) were treated with PGF2alpha (15 mg, im) 10 d apart, with controlled internal drug release (CIDR) devices containing 300 mg progesterone for 12 d (n = 18), or with intravaginal sponges containing 500 mg progesterone for 12 d (n = 18). On the day of the second PGF2alpha injection or at CIDR or sponge removal, sterile rams were placed with the ewes. Jugular blood samples were collected from the ewes at 6-h intervals until the time of ovulation, and daily for 16 d after estrus (Day 0). Plasma was harvested and stored at -20 degrees C until LH, and progesterone concentrations were determined by RIA. There was no difference (P>0.10) in time to estrus among the CIDR-, PGF2alpha- or sponge-treated ewes. All of the ewes in the CIDR group and 94.4% of the sponge treated ewes exhibited estrus by 36 h after ram introduction, while only 72.2% of PGF2alpha-treated ewes showed signs of estrus by this time (P<0.06). The time from ram introduction to ovulation was not different (P>0.10) among the CIDR-, PGF2alpha- or sponge-treated ewes. The time to the preovulatory LH surge was similar (P>0.10) among CIDR, PGF2alpha and sponge treated ewes. Progesterone levels through Day 16 after the synchronized estrus were not different (P>0.10) among treatment groups. Hair sheep ewes (n = 23) were synchronized using PGF2alpha and bred by TAI using frozen-thawed semen 48 h after the second injection. The conception rate to TAI was 2/23 (8.7%) and produced 3 ram lambs. In a subsequent trial, 17 ewes were synchronized with CIDR devices and bred by TAI using frozen-thawed semen 48 h after CIDR removal, resulting in a conception rate of 52.9% (9/17). It is possible to synchronize estrus in hair sheep using either CIDRs, sponges or PGF2alpha. Even though there were no significant differences in the timing of ovulation or the LH surge among the treatment groups, a higher conception rate was achieved in ewes synchronized with CIDR devices during the second trial. This may reflect an increase in the skill level of the TAI technician.     

Factors affecting fertility after cervical insemination with cooled semen in meat sheep

Field results of 18,328 cervical artificial inseminations (AI) with cooled semen in Rasa Aragonesa meat sheep under field conditions in north-eastern Spain AI were analyzed. Logistic regression procedures were used including fertility at AI as the dependent variable (measured by lambing, 0 or 1) and year, month of AI, farm, hours between extraction and insemination, number of ewes inseminated in a set of AI, parity, lambing-treatment interval, total number of synchronization treatment per ewe, inseminating ram and AI technician as independent factors. Previous parturitions, lambing-AI interval, month, farm, inseminating ram and technician were factors with significant impact on AI fertility. Based on the odds ratio, the likelihood of pregnancy decreased: in ewes with more than five previous parturitions (by a factor of 0.87, 0.79 and 0.66 for the 6th, 7th and ≥8 parturitions, respectively); in ewes with lambing-AI interval higher than 240 days (by a factor of 0.8); and for inseminations performed during the spring period, (March, April, May and June, 0.70, 0.76, 0.66, and 0.76, respectively). We noted a higher fertility in seven inseminating rams (odds ratios between 1.4 and 1.7) and lower in two rams (odds ratios between 0.6 and 0.7). Of the 17 AI technicians, two were related to fertilities improved by odds ratio of 1.6, and 1.30, whereas two technicians were attributed fertility rates reduced by odds ratios of 0.68 and 0.40. These findings should be taken into account to evaluate the AI technique performance and make decisions to enhance fertility results.     

Effects of heating the testes and epididymides of rams by scrotal insulation on fertility and embryonic mortality in ewes inseminated with frozen semen.

Fertilization rate and embryonic mortality were assessed in 636 ewes inseminated in each uterine horn with 50 x 10(6) frozen spermatozoa from four control rams and from four rams submitted to a moderate (1.4-2.2 degrees C), but repeated, intermittent (16 h/day for 21 consecutive days) increase in their subcutaneous scrotal temperature by means of scrotal insulation. Pregnancy was assessed twice in each ewe from concentration of progesterone in blood plasma at 17 days and by ultrasound at 65 days after insemination. No differences were observed in the pregnancy rate at 17 days between ewes inseminated with semen collected from control rams (56.0, 65.2, 66.7 and 60.3%) and from heated rams (60.6, 71.8, 63.6 and 48.2%) before or after 4, 15 and 21 days of heating, respectively. In contrast, the rate of embryonic mortality between 17 and 65 days after insemination was significantly higher at days 4, 15 and 21 in the heated rams (78.7, 78.6 and 93%) than in the control rams (55, 59 and 65.7%). These results indicate that an intermittent slight, but repeated, increase in the subcutaneous scrotal temperature could induce a significant increase in the embryonic mortality rate. As these changes were apparent on day 4 of heating, an effect must have occurred on sperm stored in the epididymis.     

Pregnancy rates in mares inseminated with 0.5 or 1 million sperm using hysteroscopic or transrectally guided deep-horn insemination techniques

Placement of sperm deep in the equine uterine horn allows fewer sperm to be inseminated while maintaining acceptable fertility, and has been promoted for use in circumstances when fertility would be expected to be low if standard insemination were used (e.g., semen from a subfertile stallion, or frozen-thawed semen). Two main techniques, transrectally guided (TRG) and hysteroscopic (HYS) insemination, have been developed for this purpose; however, there is some controversy regarding their comparative efficacy. This study was conducted to compare pregnancy rates when mares were inseminated by TRG or HYS, using sperm numbers approaching and under the minimum threshold, resulting in reduced fertility. When 1 × 10⁶ sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (10/13, 77%) and TRG (11/15, 73%). Similarly, when 0.5 × 10⁶ sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (3/15, 20%) and TRG (4/13, 31%). Combined pregnancy rates for the two treatments were 13/28 (46%) for HYS and 15/28 (54%) for TRG (P > 0.10). Pregnancy rates using a subthreshold number of sperm were not significantly affected by a deep-horn insemination technique.     

In vivo and in vitro fertilizing capacity of cryopreserved European mouflon [Ovis gmelini musimon] spermatozoa used to restore genetically rare and isolated populations

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 +/- 4.6% versus 56.0 +/- 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 +/- 4.6%) than in ram semen (62.7 +/- 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.