Sperm Localization in the Oviducts of Artificially Inseminated Dairy Cattle

Eight animals, 3 heifers and 5 primiparous cows, were artificially inseminated by intrauterine deposition of frozen-thawed semen. The insemination dose comprised 20×106 or 200 × 106 spermatozoa, frozen in French mini straws. Four animals were inseminated at fixed time interval (72 or 84 h) after cloprostenol injection. The remaining 4 animals were inseminated in spontaneous oestrus. Slaughter took place 2 or 12 h after insemination. After fixation the oviducts were cut into segments, which were serial-sectioned and stained. Six sections per segment were examined under the microscope for sperm recovery. The number of spermatozoa recovered from the oviducts varied considerably among animals. Recovery was poor (less than 50 spermatozoa) in 4 animals. Recovery was low when insemination took place in induced oestrus and with the lower sperm number (20×106). In animals in which more than 50 spermatozoa were found the distribution varied both between animals and between oviducts within the same animal. Overall, more spermatozoa were found in the lower (UTJ, isthmus and AIJ) than in the upper (ampulla) parts of the oviducts. In 3 out of 4 animals more spermatozoa were recovered from the left than from the right oviduct. Only in 1 animal were the majority of spermatozoa found in the oviduct ipsilateral to the follicle-bearing ovary.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).     

The effect of seminal sperm concentration on sperm transport in the reproductive tract of female rabbits treated with progesterone or diethylstilbestrol

Forty-two mature Baladi female rabbits were used in a randomized 3x2 factorial experiment to determine the effects of three treatments (control, progesterone injection: 2 mg/doe and DES injection: 0.1 mg/doe) and two semen sperm cell concentrations (1x10(6) and 60x10(6) sperm/0.25 ml semen on sperm transport and distribution in the female reproductive tract. The injections were given for three consecutive days after which rabbits were injected with 5 IU HCG and inseminated with 0.25 ml semen. The does were sacrificed 10 hrs after insemination and the sperm were recovered and counted from the oviducts, uterine horns, cervices and vagina. Total spermatozoa recovered was high when rabbits were inseminated with 60x10(6) sperm as compared to those inseminated with 1x10(6) sperm. When rabbits were injected with progesterone or DES, the number of sperm recovered relative to the total number of sperm inseminated was high in rabbits inseminated with 1x10(6) sperm, in comparison to those inseminated with 6x10(6) sperm. The number of sperm recovered was highest from cervix which was followed by vagina, uterus and oviducts. DES increased the number of the total sperm recovered while progesterone decreased the number as compared to control. This trend was also observed within the different segments of the reproductive tract and with groups inseminated with 1x10(6) or 60x10(6) sperm/0.25 ml semen. The effect of DES was more obvious with does inseminated with low sperm numbers. Significant correlation coefficients were found between the sperm numbers recovered in the uterus and oviducts and in the cervix and uterus of all groups of rabbits.     

Quality and fertility of cooled-shipped stallion semen at the time of insemination

Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm concentration of AI doses was 68 ± 3 x 10⁶/ml), mean total sperm count was 1.0 ± 0.1 x 10⁹, total motility averaged 83 ± 1% and morphological defects 45 ± 2%. A total of 86 mares were inseminated, overall per season-pregnancy rate in these mares was 67%. Sperm concentration significantly influenced semen motility and morphology at arrival of the shipped semen. Significant effects of month of the year on volume, sperm concentration and total sperm count of the insemination dose were found. The collection center significantly influenced all semen parameters evaluated. Semen doses used to inseminate mares that became pregnant had significantly higher total and progressive motility of spermatozoa and a significantly lower percentage of morphological semen defects than insemination doses used for mares failing to get pregnant. Results demonstrate that insemination with semen of better quality provides a higher chance to achieve pregnancy. Besides the use of stallions with good semen quality, appropriate semen processing is an important factor for satisfying results in artificial insemination with cooled-shipped horse semen.     

Improving the efficiency of sperm technologies in pigs: the value of deep intrauterine insemination

The use of AI in pigs has dramatically expanded in the last few years. New methodological advances in AI are required to serve the requirements of new sperm technologies, such as the use of low dose AI, because the use of cervical AI has a very low efficiency leading to low fertility results. One of the strategies devised to meet these requirements is the deposition of semen near the site of fertilization in the oviduct. Using deep intrauterine insemination with a specially designed catheter, a 20-fold reduction in the number of freshly and diluted inseminated spermatozoa can be achieved without decreasing farrowing rates. Moreover, an advantage of deep intrauterine insemination is the possibility of using processed, 'weaker' spermatozoa such as those that have been frozen-thawed or sex-sorted. Although deep intrauterine insemination should be of benefit to the pig industry, more investigations are needed to understand the mechanisms related to sperm colonization of the oviducts and identify the minimal sperm numbers needed to obtain maximal fertility results for processed and unprocessed boar spermatozoa.     

Identification of embryo paternity using polymorphic DNA markers to assess fertilizing capacity of spermatozoa after heterospermic insemination in boars

Differences in sperm fertilizing capacity of males often remain undetected by routine semen parameters. Heterospermic insemination with equal numbers of spermatozoa from 2 males is an accurate method for assessing differences in fertility. Use of heterospermic insemination depends on a reliable, efficient assay to identify paternity of conceptuses or offspring. In this study, polymorphic DNA markers amplified by PCR were tested to determine paternity of Day 5 to 6 embryos. The fertilizing capacity of 2 boars (A and B) with similar semen parameters was compared after homospermic (n=14 gilts) and heterospermic (n=11 gilts) insemination. Single AI's were performed under suboptimal conditions using 1 x 10(9) spermatozoa at 12 to 24 h before ovulation to prompt differences in fertilization and to stimulate sperm competition. The fertilization rate and the number of accessory spermatozoa were determined in Day 5 to 6 embryos. Using 5 different polymorphic DNA markers, paternity could be determined in 95.8% of the embryos. Boar B sired significantly (P<0.05) more offspring than Boar A after insemination with pooled semen, and this was reflected by a significantly (P<0.05) higher number of accessory spermatozoa following homospermic insemination with semen from Boar B, although fertilization rates did not differ between the 2 boars after homospermic insemination. The results suggest that the viability of spermatozoa in the female reproductive tract contributes to differences in fertility rates of males with similar in vitro sperm quality parameters. The number of accessory spermatozoa is a more sensitive measure of boar fertility than the fertilization rate. Polymorphic DNA markers are suitable for verification of parentage even at a very early stage of embryonic development.     

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.     

Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro

Experiments were carried out to study morphological changes temporally associated with in vitro fertilization (IVF) of prepubertal goat oocytes and to elucidate some of the abnormalities occurring during this process. The effects of different intervals of insemination on subsequent embryonic development were also studied. Prepubertal goat oocytes collected at slaughter were matured in TCM199 supplemented with estrous goat serum (20%), FSH (10 microg/ml), LH (10 microg/ml) and estradiol-17 beta (1 microg/ml) for 27 h at 38.5 degrees C. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (37) but with 100 microg/ml heparin. Representative oocytes were fixed every 2 to 4 h from 2 to 28 h after insemination for a study of sperm penetration, sperm head decondensation, meiotic activation, female chromosome decondensation, and male and female pronuclear formation. At the same intervals after insemination, some of the ova were co-cultured on granulosa cell monolayers for up to 9 d. Sperm penetration into the ooplasm was first observed at 4 h post insemination; decondensation of male and female chromatin and formation of male and female pronuclei occurred at 6 to 8 and 10 to 16 h after insemination, respectively. Highest proportions of oocytes were penetrated after exposure to spermatozoa for 8 h. There were no significant differences in ovum penetration after longer insemination intervals. Cleavage was first observed 24 h after insemination. Three types of abnormalities were observed. These were polyspermy, polygyny and asynchrony in the development of the female and male pronuclei, apparently due to a delay in the decondensation of the male pronucleus. Significantly higher proportions of oocytes cleaved (31.2 to 45.5%) after 20, 24 or 28 h insemination intervals than following shorter intervals of exposure to spermatozoa. However, the sperm exposure interval did not significantly affect subsequent embryonic development to the blastocyst stage. Embryos resulting from oocytes exposed to sperm cells for at least 12 h developed further than the 8-cell stage.     

Efficacy of TEPA as a “sperm label” for competitive fertilization studies in the rabbit

In this paper, the conditions necessary to use TEPA [tris (1-aziridinyl)] effectively as a label for spermatozoa in competitive fertilization are established. The fertilizing ability of rabbit spermatozoa treated with 0 and 0.8 mg TEPA/ml was compared at insemination doses of 1, 5, 20, and 40 × 10⁶ spermatozoa. Fertility was assessed by collecting ova from 64 does 48 to 52 h after insemination. TEPA blocked all but 4% of the ova from developing when 1 × 10⁶ spermatozoa were inseminated, but fertility was reduced. When 5 × 10⁶ spermatozoa were inseminated following treatment with 0, 0.6 or 1.2 mg of TEPA/ml, the fertility was 83, 74 and 50% (P<0.05), and the percentage of ova containing more than four blastomeres was 83, 11 and 5% (P<0.05), respectively. The 0.6% TEPA level was selected for a competitive fertilization trial. Equal numbers of sperm from pure Dutch-color and albino sires were combined so that either both types were untreated, only the ‘albino’ semen was treated, only the ‘Dutch’ semen was treated, or both were treated. Does were inseminated with 5 × 10⁶ sperm and allowed to kindle. The litter sizes were 5.6, 3.1, 2.7, and 0 young, and the proportion of Dutch-color progeny was 63, 97, 0 and 0%, respectively, confirming the effectiveness of TEPA as a “label”. Only one of 60 young born resulted from fertilization by a TEPA-treated spermatozoon, demonstrating that few embryos fully escape the TEPA block. Thus, the TEPA concentration and sperm numbers were established to use TEPA effectively as a label for spermatozoa in competitive fertilization studies.     

Semen backflow after insemination and its effect on fertilisation results in sows

The aim of the present study was to investigate the volume of and number of spermatozoa in semen backflow during and after insemination, and the effect of backflow on fertilisation results assessed at day 5 of pregnancy. Multiparous sows (n=140) were artificially inseminated with either (1, 3 or 6)×10⁹ mixed spermatozoa from three boars in a constant volume of 80 ml. Backflow of semen was measured three times: during insemination (M1); during the first half hour after insemination (M2); and from 0.5 h until about 2.5 h after insemination (M3). Transrectal ultrasonography was performed at intervals of 4 h to determine the time of ovulation. Sows were sacrificed at 120±0.4 h after ovulation to assess the results of fertilisation. Every sow had some backflow and the variation in volume, and number of spermatozoa within the backflow was high. The average semen backflow within 2.5 h after insemination was 70±3.4% of the volume and 25±1.4% of the spermatozoa of the inseminated dosage. The concentration of the backflow (% of the inseminated dosage) decreased with time after insemination from 65% at M1 to 40% and 26% at M2 and M3, respectively. The correlations between volume and number of spermatozoa were high: r=0.97, r=0.73 and r=0.81 in M1, M2 and M3, respectively. More than 5% of the inseminated spermatozoa in backflow during insemination affected fertilisation negatively in those sows inseminated with 1×10⁹ spermatozoa (P<0.05). Backflow after insemination had no effect on fertilisation results (P>0.05). Timing of insemination relative to ovulation and oestrus were not related to backflow during or after insemination (P>0.05). Of the sows which had backflow, those of parity 1 tended to have the highest proportion of sows with more than 5 ml backflow (47%; n=8 of 17) compared with sows from parity 2 and higher (24%; n=14 of 59) (P=0.075). It was concluded that excessive backflow of semen during insemination had a negative effect on fertilisation results when sows where inseminated with only 1×10⁹ spermatozoa. Causes of variation in backflow between sows were not clearly identifiable.     

Collection of semen and artificial insemination of alpacas

Semen collection and artificial insemination have not yet been fully developed in the alpaca. Thus, we collected semen from 7 males using a modified artificial vagina placed inside a dummy. Forty adult female alpacas, previously induced to ovulate with hCG, were artificially inseminated with fresh undiluted semen by laparoscopy or by cervix. The Chisquare test was used to determine differences in the fertility rate of the 2 insemination methods. The mean duration of copulation, semen volume, sperm concentration and the percentages of live spermatozoa and normal spermatozoa were 21.6 min, 1.9 ml. 147,500/mm(3), 69.6% and 75.9%, respectively. There were 6.7% abnormal heads, 12.3% abnormal tails and 3.8% cytoplasmic droplets. The consistency of semen was viscous and formed a coagulum. The pH was 7.2, and the semen was milky white in color. The duration of copulation was comparable to natural copulation, and semen characteristics reflected those of the natural ejaculate. The percentage of pregnancy was 68%, with no differences due to method of semen deposition (laparoscopy, 67%; cervix, 73%).     

Vaginal deposition of frozen-thawed semen in Norwegian Dairy goats: Comparison of single and double insemination with equal total number of spermatozoa

In a field trial, a total of 472 Norwegian Dairy goats showing natural estrus were artificially inseminated with frozen-thawed semen. The farmers themselves performed vaginal deposition of 400 × 10⁶ spermatozoa; one half of the does received two straws (200 × 10⁶ spermatozoa/straw) at the same time (single AI), while the other half received two straws (200 × 10⁶ spermatozoa/straw) 12 h apart (double AI). The commercially available extender Andromed^® was used for dilution. The does were housed at 15 different farms, and on average 31 does were inseminated per farm. Non return rates (NRR) and kidding rates after single insemination were 64.3% and 58.3%, respectively. Double inseminations resulted in a NRR of 62% and a kidding rate of 57%. No significant difference between single and double AI was seen in the study. This study indicates that single or double vaginal insemination with an equal total number of frozen-thawed spermatozoa (400 × 10⁶) can give acceptable fertility results in Norwegian Dairy goats. However, studies on reducing sperm numbers are called for to allow AI donor bucks to be used to their fullest potential.     

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.     

Manual semen collection from a Grevy's zebra stallion (Equus grevyi), onset of sperm production, semen characteristics, and cryopreservation of semen, with a comparison to the sperm production from a Grant's Zebra stallion (Equus burchelli boehmi)

A manual technique was used to collect representative ejaculates from an unrestrained Grevy's zebra stallion beginning at 13 mo of age to determine the onset of sperm production, to calculate the number of spermatozoa produced per ejaculate, and to determine any seasonality associated with sperm production. Spermatozoa first appeared in the ejaculate at 31 mo of age. By 48 mo of age the zebra was producing up to 40 billion spermatozoa per ejaculate. Progressive sperm motility ranged from 75 to 95%. Gel-free semen volume averaged 75 to 120 ml/ejaculate. Gel volume ranged from 0 to 1100 ml/ejaculate. Semen was frozen in 2 different extenders in 0.5-ml PVC straws. The post-thaw motility of cryopreserved spermatozoa ranged from 30 to 70%. A domestic horse mare became pregnant on the first cycle after insemination with frozen-thawed spermatozoa from this zebra. Sperm production data obtained from semen collections made on a Grant's Zebra stallion from 3 to 8 yr of age is presented for comparison of the 2 species.     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Artificial insemination in domestic cats (Felis catus)

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

Pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without Single Layer Centrifugation

A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from “normal” ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sperm quality seen in SLC-selected sperm samples compared with uncentrifuged controls in laboratory tests are reflected in an increased pregnancy rate after artificial insemination. In a multicentre study, SLC-selected sperm samples and uncentrifuged controls from eight stallions were inseminated into approximately 10 mares per treatment per stallion. Ultrasound examination was carried out approximately 16 days after insemination to detect an embryonic vesicle. The pregnancy rates per cycle were 45% for controls and 69% for SLC-selected sperm samples, which is statistically significant (P < 0.0018). Thus, the improvement in sperm quality reported previously for SLC-selected sperm samples is associated with an increase in pregnancy rate, even for ejaculates from stallions with no known fertility problem.     

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.     

Migration/sedimentation sperm selection method used in bovine in vitro fertilization: Comparison with washing/centrifugation

Sperm selection methods are usually considered for in vitro fertilization (IVF) programs. To achieve a population of viable, motile and morphologically normal spermatozoa, seminal plasma and contaminants must be removed. In this study 2 sperm selection methods were compared with regard to conventional parameters: 1) repeated washing/centrifugation, and 2) migration/sedimentation. Their effect on the fertilization of oocytes aspired from ovaries of slaughtered cows was determined. Frozen-thawed semen was used for all assays (n = 8). The sperm concentration was adjusted to 1.0 × 10⁶ cells/ml for in vitro insemination. Selected oocytecumulus complexes were matured in vitro for 24 h and were co-incubated with spermatozoa prepared by the 2 different methods. Presumptive zygotes were co-cultered in TCM-199. The percentage of motile, viable and morphologically normal spermatozoa was markedly higher (p < 0.05) with the migration-sedimentation method. Triple stain showed that the integrity of the acrosome was significantly better maintained after migration/sedimentation (68.3%) than after washing/centrifugation (53.2%; p < 0.05). In accordance with these results, a high fertilisation rate was achieved after application of the migration/sedimentation method (92.5 vs 69.1%;p < 0.05). It is concluded, that this method is very promising for obtaining spermatozoa with optimal fertilization capacity.     

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.