Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen.

Semen cryopreservation and artificial insemination (AI) are potentially valuable methods for supporting the breeding management of endangered species like the Asian elephant. Cryopreservation of Asian elephant semen has however proven problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. In this study, nine ejaculates from three adult bulls were used to compare the influence of extender (TEST versus INRA96®) and penetrating cryoprotectants (3% glycerol, 5% glycerol and 4% methylformamide) on post-thaw semen quality. We demonstrate that not only the freezing process, but also the quality of the semen before freezing, significantly influences the freezability of Asian elephant semen. Pre-freeze motility, viability, semen volume, semen pH, sperm concentration and the incidence of sperm mid-piece and tail abnormalities all significantly (p<0.05) affected post-thaw semen quality. While extender and cryoprotectant did not significantly affect any of the above semen quality parameters post-thaw, the skim-milk based extender (INRA96®) preserved DNA integrity better (p<0.05) than the egg yolk extender (TEST). Considerable between-ejaculate variation in all post-thaw semen quality parameters was also noted. It is concluded that strict criteria for semen quality is essential for the selection of Asian elephant bull ejaculates suitable for cryopreservation; stricter initial selection should improve the mean post-thaw quality.

Relationship of flow cytometric sperm integrity assessments with boar fertility performance under optimized field conditions.

The number of intact and functional spermatozoa in semen can be assessed with flow cytometry and is believed to relate to male fertility. The aim of this study was to examine whether currently used sperm integrity assessments with flow cytometry correlate with field fertility data obtained for boar semen. For this purpose, 20 boars were followed for a 20-wk period (with a total average production of 33 ejaculates per boar) and the obtained fertility results (farrowing rate and number of piglets born) of commercial artificial insemination doses made from these ejaculates were recorded. Fertility results were corrected for farm, sow, boar, and semen-related parameters. From the same semen samples, sperm cell integrity was assessed with respect to DNA and to membrane integrity, acrosome intactness and responsiveness, and mitochondrial potential using established flow cytometric assays. This was done on freshly produced semen and on semen stored for up to 15 d. Remarkably, none of the individual membrane integrity variables was significantly related to fertility results. In contrast, the amount of DNA damage as assessed at 7 to 10 d and at 14 to 15 d of semen storage related to farrowing rate (P = 0.0400) and total number of piglets born (P = 0.0310), respectively. Therefore, the degree of DNA damage in stored boar semen samples may be a useful factor to evaluate semen as an indicator for litter size and farrowing rate.

The value of microscopic semen motility assessment at collection for a commercial artificial insemination center, a retrospective study on factors explaining variation in pig fertility.

This study was conducted to evaluate the relationship between boar and semen related parameters and the variation in field fertility results. In 8 years time semen insemination doses from 110 186 ejaculates of 7429 boars were merged to fertility parameters of inseminations of 165 000 sows and these records were used for analysis. From all ejaculates boar and semen related data were recorded at the artificial insemination (AI) centers. Fertility parameters, such as farrowing rate (FR), ranging between 80.0% and 84.0%, and the total number of piglets born (TNB), ranging between 12.7 and 13.1, were recorded and from these the least square means per ejaculate were calculated. Only 5.9% of the total variation in FR was due to boar and semen variability of which 21% (P = 0.0001) was explained by genetic line of the boar, 11% (P = 0.047) was explained by laboratory technician, and 7% (P = 0.037) was explained by the AI center. For TNB the total variation was 6.6% boar and semen related of which 28% (P < 0.0001) was explained by genetic line of the boar and 7% (P = 0.011) was explained by the AI center. Only 4% of the boar and semen related variation was caused by sperm motility (microscopically assessed at collection, ranging from 60% to 90%). Other variation in FR and TNB was explained by management and semen related parameters (age of boar, 3%; P = 0.009; and 8%; P = 0.031, respectively), days between ejaculations (1%; P < 0.0001 of FR), number of cells in ejaculate (1%; P = 0.042 of TNB), year (9%; P = 0.032), and 13%; P = 0.0001, respectively), and month (11%; P = 0.0001; and 5%; P = 0.0001, respectively). Although semen motility is considered an important parameter to validate the quality of the ejaculate processed, it only minimally relates to fertility results under the current Dutch AI practice. Other boar and semen related parameters, like genetic line of the boar, are more relevant factors to select boars for AI purposes.

Application of computer-assisted semen analysis to explain variations in pig fertility.

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P < 0.05) effects of progressive motility, velocity curvilinear, and beat cross frequency on farrowing rate (FR). Total motility, velocity average path, velocity straight line, and amplitude of lateral head displacement affected (P < 0.05) total number of piglets born (TNB). Boar- and semen-related parameters explained 5.3% of the variation in FR and 5.9% of the variation in TNB. Motility parameters, measured by CASA, explained 9% of the boar- and semen-related variation in FR and 10% of the boar- and semen-related variation in TNB. Individual boar and genetic line of the boar affected (P < 0.0001) the variation in FR and TNB. No differences (P > 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination.

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.

The roles of the epididymis and prostasomes in the attainment of fertilizing capacity by stallion sperm.

The epididymis is a long, tightly coiled tube within the lumen of which sperm matures. Sperm maturation involves morphological and biochemical changes in the sperm plasma membrane in response to epididymal secretions and their various proteins. Some of these proteins become outer membrane components while others become integral membrane proteins; transfer of some proteins to the sperm plasma membrane may be mediated by epididymosomes. Nevertheless, the molecular pathways by which spermatozoa acquire fertilizing capacity during their transit through the epididymis remain ambiguous. In a recent study of stallion epididymal sperm, we found that sperm harvested from different parts of the epididymis (caput, corpus and cauda) had a varying, but generally poor, ability to undergo the acrosome reaction in vitro. At ejaculation, however, sperm mix with seminal plasma which contains various components, including the small membranous vesicles known as prostasomes, that may enable the sperm to undergo physiological activation. Seminal plasma components may have a 'washing' effect and help to remove 'de-capacitation' factors that coat the sperm during storage in the cauda epididymis; alternatively seminal plasma and prostasomes may contain factors that more directly promote sperm activation. This article reviews current information on the roles of epididymal and accessory gland fluids on the acquisition of fertilizing capacity by stallion sperm.

Harnessing the biology of the oviduct for the benefit of artificial insemination.

Spermatozoa fulfil a single role, namely achieving syngamy by transporting the haploid genome to their counterpart gamete, the oocyte. Simple as this may seem, it is fraught with many difficulties, especially in the face of biological processes that enable females to select spermatozoa after they have mated multiply with several males. Conversely, the female reproductive tract sequesters a privileged sperm subpopulation in the oviductal isthmus for variable periods of time, releasing them when the time is opportune for fertilisation. Recent studies of sperm transport in the female reproductive tract suggest that these phenomena involve signalling dialogues between spermatozoa and the female reproductive tract environment. Opportunities for mutual signalling are immense but have received relatively little attention. The oviduct is an organ of crucial significance in modulating sperm function and may be one of the most important sites for determining many aspects of sperm selection and competition. The oviductal environment possesses the potential for enhancing sperm survival, suppressing and activating sperm motility as required, and responds to the arrival of spermatozoa by producing novel proteins. While the biological nature of the sperm-oviduct dialogue is interesting for its own sake, the mechanisms that govern these processes offer opportunities for the improvement of artificial insemination procedures. If oviductal proteins enhance sperm survival, they offer opportunities for the development of long-life semen diluents. Conversely, if we understood the basis of sperm selection we may be able to concentrate on identifying and using only the best sperm subpopulations for improved animal breeding efficiency.

Effects of histamine on gizzard erosions and on the activity of selected enzymes in chickens.

Dose- (5, 10 and 15 mg/kg body mass) and time-dependent (2, 6, 12 and 24 h after treatment) effects of histamine on gizzard lesions and serum AST, ALT and CK activities of chickens are reported. Morphometric results and histopathological examination revealed that the most effective histamine dose which induced severe gizzard lesions was 10 mg/kg b.m., especially 2, 6 and 12 h after administration. No difference from the control values was found after 24 h. That dose also induced an elevation of serum enzyme (AST, ALT, CK) activities, which was most expressed for the activity of ALT.