Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare

Our objective was to determine whether the concentration of cooled sperm inseminated influenced sperm transport and intensity of the uterine inflammatory reaction 2, 4 and 24h after insemination. Experimental subjects were 189 estrous mares with a dominant follicle > or =35 mm in diameter and no bacterial growth or neutrophils detected in uterine smears. Each mare was randomly assigned to receive one of the following intrauterine treatments (volume, 20 mL): insemination with 5x10(6) mL(-1) or 25x10(6) mL(-1) or 50x10(6) mL(-1) sperm diluted in 3 mL seminal plasma (SP) and 17 mL skim milk; seminal plasma or skim milk extender. Mares in a control group received no intrauterine treatment. Mares were slaughtered 2, 4 or 24h after insemination or infusion. Oviducts were separated from the uterus, and uterus and oviducts were then flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected for further histopathological examination. The grade of uterine fibrosis and the amount of neutrophils in the stratum compactum were evaluated. A sample of each tubal flushing was examined for sperm count, and a sample of each uterine flushing was examined for PMN count. It was concluded that compounds in the insemination dose provoked a uterine inflammatory response, which was more rapid and intense as sperm concentration increased. In contrast, sperm transport through 4h after insemination was not influenced by sperm concentration.     

Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination.

An artificial insemination dose for mares consisting of 500 million progressively motile spermatozoa is considered "standard" by most clinicians. However, little information is available directly comparing pregnancy outcome among methods of evaluating and selecting spermatozoa for insemination. The objective of this study was to determine if the method of spermatozoal evaluation and selection influences fertility as measured by pregnancy outcome. Mares were inseminated with 100 or 500 million spermatozoa that were selected for progressive motility, normal morphology, hypoosmotic swelling or absolute number regardless for evaluation method or quality. Thirty-two breeding cycles were tested for each treatment group and at each spermatozoal dose. Pregnancy outcomes were 44 and 41%, 55 and 41%, 39 and 31%, and 45 and 41%, for the 100 and 500 million progressively motile, morphologically normal, hypoosmotic swelling positive and absolute number treatment groups, respectively. Pregnancy outcome did not differ among methods of spermatozoal evaluation and selection for artificial insemination in the 100 (P=0.52) or 500 (P=0.78) million spermatozoa groups. Also the total number of spermatozoa and the absolute number of progressively motile, morphologically normal or hypoosmotic swelling positive spermatozoa inseminated, were not closely associated with pregnancy outcome in the 100 (P=0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P=0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa groups. In this study, we found that the method of spermatozoal evaluation did not offer an advantage for pregnancy when used to select spermatozoa for insemination at the doses tested. These results were surprising, as we expected there would be differences among the evaluation methods. Instead, we found that evaluating spermatozoa offered no advantage for pregnancy over simply inseminating with a specified number of spermatozoa not selected for any particular characteristic under the conditions of our experiment.     

Pregnancy rates in mares following hysteroscopic or transrectally-guided insemination with low sperm numbers at the utero-tubal papilla

This study was conducted to evaluate two methods for insemination of a low number of sperm in the tip of the uterine horn, and to determine whether prebreeding intrauterine treatment with prostaglandin E(2) would improve pregnancy rates. Estrus was synchronized in 36 fertile Quarter Horse and Thoroughbred broodmares. When a dominant follicle >or=33 mm diameter was present, mares were treated with 2500 units hCG intravenously and were assigned to one of four treatment groups for insemination with five million total sperm in 200 microl extender the next day as follows: (1) Group PGE-HYS (n=9): 0.25mg PGE(2) in 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to hysteroscopic-guided inseminate placement onto the oviductal papilla; (2) Group SAL-HYS (n=9): 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to hysteroscopic-guided inseminate placement onto the oviductal papilla; (3) Group PGE-PIP (n=9): 0.25mg PGE(2) in 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to transrectally-guided pipette placement of the inseminate into the tip of the uterine horn; and (4) Group SAL-PIP (n=9): 1 ml 0.9% NaCl solution infused into the tip of the uterine horn ipsilateral to the dominant follicle 2h prior to transrectally-guided pipette placement of inseminate into the tip of the uterine horn. Mares in estrus were evaluated daily by transrectal ultrasonography to monitor follicular status and confirm ovulation. If mares had not ovulated within 2 days of insemination, the assigned treatment was repeated. Pregnancy status was evaluated by transrectal ultrasonography 12-14 days postovulation, and pregnancy rates were compared.No interaction between prebreeding treatment (SAL:PGE) and insemination protocol (HYS:PIP) on pregnancy rates occurred (P>0.10). Pregnancy rates did not differ between mares inseminated by HYS (12/18; 67%) or PIP (10/18; 56%) (P>0.10). Pregnancy rates did not differ between mares treated prior to breeding with PGE (11/18; 61%) or SAL (11/18; 61%) (P=1.00). In summary, satisfactory pregnancy rates were obtained when a low number of sperm were either placed directly onto the oviductal papilla using hysteroscopy or placed in the tip of the uterine horn using a transrectally-guided uterine pipette. Infusion of 0.25mg PGE(2) in the tip of the uterine horn 2h prior to insemination did not improve pregnancy rates.     

Effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method of semen (fresh, frozen-thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50 x 10(6) or 300 x 10(6) progressively motile sperm (pms) for fresh semen and 100 or 800 x 10(6) frozen-thawed sperm with >35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG administration when fresh semen was used, or 30 h for frozen-thawed semen. Differences in pregnancy rates between treatment groups were analyzed by Chi-squared test, and for most relevant factors (insemination technique, mare, semen, and stallion) expectation values and confidence intervals were calculated using multivariate logistic models. Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects (P > 0.05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between insemination technique, semen parameters, and mares did have significant effects (P < 0.05). In problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI (16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares, hysteroscopic AI with fresh semen gave significantly (P < 0.05) better pregnancy rates than uterine body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results demonstrate that for problem mares, conventional insemination into the uterine body appears to be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be expected by hysteroscopic insemination.     

Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of functional sperm

Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.     

Assessment of a new utero-tubal junction insemination device in dairy cattle

A new artificial insemination device for semen deposition near the utero-tubal junction in cattle (Ghent device) has been developed at the Ghent University (Belgium). In this study, the effect of the new insemination device on sperm quality was evaluated. Moreover, in a field trial 4064 dairy cows were inseminated by 12 inseminators to examine the efficacy of the device under field conditions.The Ghent device is a disposable plastic catheter which can easily follow the curvature of the uterine horns and thus reach the utero-tubal junction (UTJ). After expulsion of the inseminate with 0.7 or 1.7 ml of air, 19.0% of the insemination dose remained in the insemination catheter. Sperm loss can be diminished to 9.0% of the original insemination dose when the insemination catheter is flushed with 0.1 ml of air, followed by 0.6 ml of physiological saline solution. No toxic effect of the insemination catheter on sperm quality or fertilizing capacity was found. In the field trial, sperm were inseminated in dairy cattle which were divided in three groups. The first group was inseminated in the uterine body with the conventional insemination device, the second group in the uterine body with the Ghent device, and the third group in the tip of both uterine horns with the Ghent device. Each insemination was performed with 10 x 10(6) to 15 x 10(6) frozen-thawed spermatozoa. The pregnancy rates (PRs) were significantly affected by the insemination technique (P = 0.02), by the inseminator (P = 0.01), by heifer or cow (P < 0.01), and by the insemination number (P < 0.01). Pregnancy rates obtained with the conventional insemination device (57.6%) were significantly better than those obtained with the Ghent device in the uterine body (52.7%) (P < 0.01), but did not differ significantly from those obtained after deep insemination into both uterine horns (53.8%) (P = 0.27). It can be concluded that the Ghent device is suitable for utero-tubal junction insemination of dairy cattle under field conditions. Whether the Ghent device is also suitable for insemination with lower insemination doses is at present under investigation.     

Timing of insemination using sex-sorted sperm in embryo production with Bos indicus and Bos taurus superovulated donors

Two experiments were designed to evaluate the effect of different insemination times (12 and 24h or 18 and 30h) and different types of semen (sex-sorted or non-sorted sperm) on embryo production in Nelore (Bos indicus) and Holstein (Bos taurus) superstimulated donors. In the first experiment, hormonal superstimulation of ovarian follicular development in Nelore donors (n=71) was performed in randomly allocated animals to one of the three treatment groups, and they were inseminated at 12 and 24h after an ovulatory stimulus with pLH treatment was applied, either with sex-sorted (4.2×10(6) sperm/insemination; S12/24; n=17) or non-sorted sperm (20×10(6) sperm/insemination; NS12/24; n=18), or they were inseminated at 18 and 30h using sex-sorted sperm (4.2×10(6) sperm/insemination; S18/30; n=19). A greater number of transferable embryos were found when sex-sorted sperm was used to inseminate the animals at 18 and 30h (4.5±3.0) compared to insemination at 12 and 24h (2.4±1.8; P<0.001). However, a greater embryo production (6.8±2.6) was obtained with non-sorted sperm. In the second experiment, the same insemination times and semen types were used in lactating high-production Holstein cows (n=12). A crossover design was employed in this trial. A lesser embryo production (P=0.007) was found in Holstein donors that were inseminated using sex-sorted sperm at 12 and 24h (4.6±3.0) compared to non-sorted sperm (8.7±2.8). However, intermediate results were obtained when the inseminations with sex-sorted sperm were performed at 18 and 30h (6.4±3.1). These results supported the current hypothesis that it is possible to improve embryo production using sex-sorted sperm in B. indicus and B. taurus superstimulated donors when the inseminations are performed near the same time as time-synchronized ovulations. However, the embryo production for timed artificial insemination (TAI) with sex-sorted sperm was still less than the production with non-sorted sperm.     

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10⁸ sperm; PS: 13.15 ± 2.39 nmol TBARS/10⁸ sperm; S: 13.20 ± 2.18 nmol TBARS/10⁸ sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10⁸ sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10⁸ sperm, PS: 25.61 ± 5.85 nmol TBARS/10⁸ sperm, S: 19.16 ± 3.28 nmol TBARS/10⁸ sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10⁸ sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.     

Effect of intrauterine treatment with prostaglandin E2 prior to insemination of mares in the uterine horn or body

Two trials were conducted to investigate the effects of intrauterine infusion of PGE2 and uterine horn insemination on pregnancy rates in mares achieved by breeding with a suboptimal number of normal spermatozoa. Estrus was synchronized and mares were teased daily with a stallion to detect estrus. Mares in estrus were examined by transrectal palpation and ultrasonography to monitor follicular status. On the first day a 35-mm diameter follicle was present, hCG (1500 IU, iv) was administered and the mares were bred the next day. Mares (Trial 1, n = 34; Trial 2, n = 28) were inseminated with 25 million total spermatozoa from either a stallion with good semen quality (Trial 1) or poor semen quality (Trial 2). In each trial, mares were assigned to 1 of 4 treatment groups as follows: Group PGE-HI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; Group PGE-BI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body; Group SAL-HI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; or Group SAL-BI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body. After breeding, mares were examined daily by transrectal ultrasonography to confirm ovulation, and were re-examined 14 to 16 d after ovulation for pregnancy status. Data were analyzed by Chi-square. Overall pregnancy rates were 59% for stallion 1 and 29% for stallion 2. Group pregnancy rates did not differ for mares bred by either stallion (P > 0.10). Pregnancy rates were not altered by horn insemination for either stallion (P > 0.10). Intrauterine infusion of PGE2 improved pregnancy rate in mares bred by the stallion with good quality semen (P < 0.05), but did not alter pregnancy rate in mares bred by the stallion with poor quality semen (P > 0.10). Further research is warranted to determine if intrauterine infusion of PGE2 will enhance spermatozoal colonization of the oviduct and pregnancy rates in mares, and if PGE-treatment will improve pregnancy rates achieved by subfertile stallions.     

Low dose insemination of mares using non-sorted and sex-sorted sperm.

Mares are generally inseminated with 500 million progressively motile fresh sperm and approximately 1 billion total sperms that have been cooled or frozen. Development of techniques for low dose insemination would allow one to increase the number of mares that could be bred, utilize stallions with poor semen quality, extend the use of frozen semen, breed mares with sexed semen and perhaps reduce the incidence of post-breeding endometritis. Three low dose insemination techniques that have been reported include: surgical oviductal insemination, deep uterine insemination and hysteroscopic insemination.Insemination techniques: McCue et al. [J. Reprod. Fert. 56 (Suppl.) (2000) 499] reported a 21% pregnancy rate for mares inseminated with 50,000 sperms into the fimbria of the oviduct.Two methods have been reported for deep uterine insemination. In the study of Buchanan et al. [Theriogenology 53 (2000) 1333], a flexible catheter was inserted into the uterine horn ipsilateral to the corpus luteum. The position of the catheter was verified by ultrasound. Insemination of 25 million or 5 million spermatozoa resulted in pregnancy rates of 53 and 35%, respectively. Rigby et al. [Proceedings of 3rd International Symposium on Stallion Reproduction (2001) 49] reported a pregnancy rate of 50% with deep uterine insemination. In their experiment, the flexible catheter was guided into position by rectal manipulation.More studies have reported the results of using hysteroscopic insemination. With this technique, a low number of spermatozoa are placed into or on the uterotubal junction. Manning et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 84] reported a 22% pregnancy rate when 1 million spermatozoa were inserted into the oviduct via the uterotubal junction. Vazquez et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 82] reported a 33% pregnancy rate when 3.8 million spermatozoa were placed on the uterotubal junction. Recently, Morris et al. [J. Reprod. Fert. 188 (2000) 95] utilized the hysteroscopic insemination technique to deposit various numbers of spermatozoa on the uterotubal junction. They reported pregnancy rates of 29, 64, 75 and 60% when 0.5, 1, 5 and 10 million spermatozoa, respectively, were placed on the uterotubal junction.Insemination of sex-sorted spermatozoa: One of the major reasons for low dose insemination is insemination of X- or Y-chromosome-bearing sperm. Through the use of flow cytometry, spermatozoa can be accurately separated into X- or Y-bearing chromosomes. Unfortunately, only 15 million sperms can be sorted per hour. At that rate, it would take several days to sort an insemination dose containing 800 million to 1 billion spermatozoa. Thus, low dose insemination is essential for utilization of sexed sperm. Lindsey [Hysteroscopic insemination with low numbers of fresh and cryopreserved flow-sorted stallion spermatozoa, M.S. Thesis, Colorado State University, Fort Collins, CO, USA, 2000] utilized either deep uterine insemination or hysteroscopic insemination to compare pregnancy rates of mares inseminated with sorted, fresh stallion sperm to those inseminated with non-sorted, fresh stallion sperm. Hysteroscopic insemination resulted in more pregnancies than ultrasound-guided deep uterine insemination. Pregnancy rate was similar for mares bred with either non-sorted or sex-sorted spermatozoa.In a subsequent study, Lindsey et al. [Proceedings of 5th International Symposium on Equine Embryo Transfer (2000) 13] determined if insemination of flow-sorted spermatozoa adversely affected pregnancy rates and whether freezing sex-sorted spermatozoa would result in pregnancies. Mares were assigned to one of four groups: group 1 was inseminated with 5 million non-sorted sperms using hysteroscopic insemination; group 2 was inseminated with 5 million sex-sorted sperms using hysteroscopic insemination; group 3 was inseminated with non-sorted, frozen-thawed sperm; and group 4 was inseminated with sex-sorted frozen sperm. Pregnancy rates were similar for mares inseminated with non-sorted fresh sperm, sex-sorted fresh sperm and non-sorted frozen sperm (40, 37.5 and 37.5%, respectively). Pregnancy rates were reduced dramatically for those inseminated with sex-sorted, frozen-thawed sperm (2 out of 15, 13%). These studies demonstrated that hysteroscopic insemination is a practical and useful technique for obtaining pregnancies with low numbers of fresh spermatozoa or low numbers of frozen-thawed spermatozoa. Further studies are needed to determine if this technique can be used to obtain pregnancies from stallions with poor semen quality. In addition, further studies are needed to develop techniques of freezing sex-sorted spermatozoa.     

Effect of a deep uterine insemination on spermatozoal accessibility to the ovum in cattle: a competitive insemination study

A competitive insemination study was conducted to determine the effect of a deep uterine insemination on accessory sperm number per embryo in cattle. Cryopreserved semen of a fertile bull characterized by spermatozoa with a semi-flattened region of the anterior sperm head (marked bull) was matched with cryopreserved semen from an unmarked bull having spermatozoa with a conventional head shape. Using 0.25-mL French straws and a side delivery embryo transfer device, deep uterine insemination (0.125 mL deposited in each horn) was performed 2 cm from the uterotubal junction. Immediately after, the uterine body was artificially inseminated using semen (0.25 mL) from an alternate bull and a conventional insemination device. The complete dose (both inseminations) was 50x10(6) total sperm cells consisting of an equal number of spermatozoa from each bull. Single ovulating cows (n = 95) were inseminated at random with either the unmarked semen in the uterine body and marked semen in the uterine horn, or the unmarked semen in the uterine horn and marked semen in the uterine body. Sixty-one embryos(ova) were recovered nonsurgically 6 d post insemination, of which 40 were fertilized and contained accessory spermatozoa. The ratio and total number of accessory spermatozoa recovered was different among treatments: 62:38 (326) for the unmarked semen in the uterine body and marked semen in the uterine horn, and 72:28 (454) for the unmarked semen in the uterine horn and marked semen in the uterine body (P<0.05). Deep uterine insemination using this semen in a split dose and a side delivery device favors accessibility of spermatozoa to the ovum compared with conventional uterine body insemination.     

Post-coital sperm recovery and cryopreservation in the Sumatran rhinoceros (Dicerorhinus sumatrensis) and application to gamete rescue in the African black rhinoceros (Diceros bicornis)

Sumatran rhinoceros (Dicerorhinus sumatrensis) sperm samples were collected from a post-copulatory female and characterized to determine their potential for sperm preservation and future use in artificial insemination. Five samples of acceptable quality from one male were used to compare the effect of two cryoprotectants (glycerol and dimethyl sulfoxide (DMSO)) and two post-thaw protocols (untreated and glass wool column) on sperm quality. The percentage of motile spermatozoa, sperm motility index (0-100) and sperm morphology were evaluated subjectively, and viability and acrosomal status were assessed using fluorescent markers. Evaluations of frozen-thawed spermatozoa were performed over a 6 h incubation interval. Post-coital semen samples (n = 5; 104.0 +/- 9.1 ml; 2.5 +/- 0.8 x 10(9) total spermatozoa; mean +/- SEM) exhibited a sperm motility index of 56.7 +/- 3.3, and contained 40.2 +/- 6.3%, 72.0 +/- 3.2% and 79.8 +/- 6.5% normal, viable and acrosome-intact spermatozoa, respectively. Glycerol and DMSO were equally effective as cryoprotectants and, regardless of post-thaw protocol, samples retained greater than 80% of all pre-freeze characteristic values. Processing semen samples through glass wool yielded higher quality samples, but only half the total number of motile spermatozoa compared with untreated samples. High values for pre-freeze sperm characteristics were also maintained after cryopreservation of epididymal spermatozoa from one black rhinoceros (Diceros bicornis) using the same protocol. In summary, Sumatran rhinoceros spermatozoa of moderate quality can be collected from post-copulatory females. Rhinoceros sperm samples show only slight reductions in quality after cryopreservation and thawing and have potential for use in artificial insemination.     

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

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

Factors influencing the success of transcervical insemination in Merino ewes

The experiments described examined the effects of a number of factors on the level of uterine insemination achieved in Merino ewes by a transcervical insemination technique (Guelph system for transcervical artificial insemination; GST-AI). Cervical penetration rate is an important limitation to the use of such methods in Merinos. Simulated insemination was performed to estimate the proportion of ewes in which a pipette could be passed through the cervix to the uterus. In Experiment 1, cervical penetration rate (n = 14 to 30) was unaffected by an increase in postpartum interval at AI from 12 to 26 wk. The results of cervical penetration for individual ewes were found to be repeatable (P < 0.05). Experiment 2 (197 ewes) revealed a clear effect of ewe parity on penetration rates in hormonally synchronized ewes during the nonbreeding season (P < 0.05). In Experiment 3, estrus synchronization using progestagen (n = 51) or prostaglandin (n = 50) did not affect penetration rate. The penetration rate was slightly higher in the naturally cycling ewes, but the difference was not significant. Comparison of ewes from Experiments 2 and 3 suggests the possibility of a major effect of stage of the breeding season on the penetration rate (P < 0.05). It is concluded that ewe selection and management techniques may be used to increase the proportion of transcervical insemination attempts resulting in uterine insemination. However, fertility testing will be required to determine whether such improvements translate into correspondingly increased pregnancy rates.     

Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions

The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10⁶ unsorted sperm per oviduct; (2) single LI with 0.5 × 10⁶ sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10⁶ sex-sorted sperm per oviduct and 0.5 × 10⁶ sex-sorted sperm per uterine horn. The farrowing rates were lower (P < 0.05) in sows inseminated with sex-sorted sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10⁶ sperm per oviduct and 1 × 10⁶ sperm per uterine horn; or (2) 1 × 10⁶ sperm per oviduct and 2 × 10⁶ sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10⁶ sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.     

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.     

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.     

New developments in low-dose insemination technology

New nonsurgical procedures for inseminating swine with a low number of spermatozoa have been developed and/or evaluated over the last few years. These procedures allow the deposition of the insemination dose into the uterine body (post-cervical insemination) or directly into the uterine horn (deep intrauterine insemination). With the use of the post-cervical insemination, a threefold reduction in the number of fresh sperm has been successfully used to achieve pregnancy. Using deep intrauterine insemination (DUI), up to a 20-fold reduction in the number of fresh spermatozoa or a sixfold reduction in the number of frozen/thawed spermatozoa can be achieved, with reproductive performance very similar to that obtained after standard AI. Complementing these nonsurgical insemination techniques, a new procedure for depositing spermatozoa into the oviduct by laparoscopy has been recently described. This laparoscopic technique has proven to be applicable to diluted and sex-sorted spermatozoa. The development of new insemination procedures will help achieve more efficient application of currently available sperm technologies. Using appropriate insemination procedures, it is now feasible to achieve high fertility rates with cooled, frozen-thawed, or sex-sorted semen.     

Effect of whole blood and serum on bovine sperm quality and in vitro fertilization capacity

Under physiologic conditions, low concentrations of blood may be present in the uterine fluid of the estrous cow at the moment of insemination. To decrease the insemination dose and to obtain good insemination results with less fertile semen, more invasive insemination methods such as utero tubal junction (UTJ) insemination can be used. More invasive insemination methods increase the risk of damaging the hyperemic endometrium, with blood in the uterine fluid as result. In this study, the effect of 0, 0.15 and 1.5% whole blood and serum on bovine sperm quality and in vitro fertilizing capacity was evaluated. Sperm quality as assessed by total motility, progressive motility, membrane integrity and acrosomal status was not affected by the presence of blood or serum (P > 0.05). However, the in vitro fertilizing capacity decreased with increasing concentrations of blood and serum (P < 0.01). The rate of polyspermy increased with increasing concentrations of serum (P < 0.01), but not with increasing concentrations of blood (P = 0.30). In conclusion, no immediate effect of blood and serum was visible on several sperm quality parameters, except for an increased prevalence of head to head agglutination (HHA). However, blood and serum did have a negative effect on in vitro fertilizing capacity.     

Embryo production with sex-sorted semen in superovulated dairy heifers and cows

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15–hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30–45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8–14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.