In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Department of Animal Pathology (Animal Health), University of Leon, Leon, Spain.
Reproduction in Domestic Animals (Impact Factor: 1.52). 05/2006; 41(2):153-61. DOI: 10.1111/j.1439-0531.2006.00659.x
Source: PubMed


Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.

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    ABSTRACT: Frozen-thawed boar sperm holds the potential to have an impact on the future of the swine industry. Utilization of this technology could improve a swine producer’s ability to access top-tier genetics from around the world, to improve efficiency, profitability, and the quality of product to meet consumer demands. Effective application of frozen-thawed sperm can help reduce the potential risk associated with devastating economic loss due to the spread of disease. Frozen storage of boar sperm also provides a safeguard in the event of disease outbreaks, as genetic material from paternal lines can be preserved and banked for repopulation purposes. Historically these benefits have been masked by reduction in fertility measures such as litter size. The reduced fertility results from the damage sustained by the sperm cell during cryopreservation. However, increased understanding of this damage has lead to improved cryopreservation methods, ultimately increasing post-thaw viability and fertility. Enhancements in breeding technology have also resulted in a better understanding of the AI methods required to achieve acceptable farrowing rates and litter size. Fertility following AI with frozen-thawed sperm is approaching that of liquid stored sperm, and producers may soon reap the benefits of this technology. This thesis will outline the current swine industry, opportunities for utilizing frozen-thawed sperm, the main components of sperm, why they are susceptible to damage, and current freezing and breeding practices. Objective 1 was to develop a cryopreservation protocol for our lab that resulted in consistent post-thaw motility ( ≥ 40%) that would eventually be used by Illinois boar studs for domestic and international sale of frozen sperm. Evaluation with both manual microscopy and CASA methods were conducted to verify quality. A preliminary breeding trial was then conducted to test the fertility of sperm frozen with this method. There were 41 ejaculates from 23 boars used for freezing. Sperm were frozen at 1.4x109 sperm/mL, averaging 55.61.1% (meanSE) motility, following thaw. The samples assessed were not different (P>0.05) in motility when compared with manual or CASA systems, and results were most reliable at a 1:40 sperm dilution. In the preliminary breeding trial, gilts (n=14) were inseminated with either a single (n=10) or double (n=4) AI using 1, 2, or 4x109 motile, frozen-thawed sperm. Overall, the resulting pregnancy rates averaged 71.4% and numbers of normal fetuses per litter averaged 15.51.3 per litter. A feasibility study for freezing cost per ejaculate was estimated at $275/ejaculate or $11/dose of frozen-thawed semen at standard doses of 5x109 total frozen-thawed sperm. This cost estimate did not include genetic value, fixed equipment costs, depreciation, or variable lab space fees. Objective 2 focused on the proper methods for breeding with frozen-thawed boar sperm to achieve fertility. Our hypothesis was that increased numbers of inseminations and increased numbers of motile frozen-thawed sperm would improve pregnancy rate and litter size. Results showed acceptable fertility at high sperm numbers, but also the optimal method for insemination with the lowest dose tested. Gilts (n=111) responded to synchronization methods and were bred with 1, 2, or 4x109 motile frozen-thawed sperm from six boars using a single AI at 32 h, or a double AI, with the first AI at 24 and 32 h following estrus. Ultrasound was conducted at 12 h intervals to estimate the time of ovulation. On day 32 of gestation, overall pregnancy rate (73%) and number of normal fetuses per litter (10.80.5) across all treatments did not differ, and were not affected by number of motile sperm, or the interaction of number of motile sperm and number of inseminations. However, the number of inseminations tended to affect (P=0.14) the number of normal fetuses. Litter size increased with a double AI compared to single AI. Multiple inseminations helped to allow insemination to occur close to ovulation in response to variation in the time of ovulation. Both pregnancy rate and number of normal fetuses were greater when the time of the AI at 32 h occurred closer to the estimated time of ovulation (P<0.05). In addition, other factors such as presence of an abnormal ovary at day 30 decreased (P<0.001) pregnancy rate, while boar affected number of normal fetuses (P<0.01). Analysis of our data using a fertility index revealed doses of 2x109 motile sperm with multiple AI can achieve acceptable fertility with use of less sperm, when compared to AI using 4x109 motile sperm. The methods described here will investigate the potential for improved fertility when using frozen-thawed sperm, while accounting for variation in time of ovulation.
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    ABSTRACT: In this study we demonstrate, in the frozen state, the architecture of frozen boar spermatozoa collected from the sperm-rich fraction of ejaculates (n=13) from four fertile boars packed and split-frozen in medium-straws (MS) and MiniFlatPacks (MFP), cross-sectioned in the frozen state and evaluated by image analysis on images obtained by use of cryo-scanning electron microscopy (Cryo-SEM). The tested hypothesis was that the degree of in situ dehydration and levels of homogeneity of boar semen either frozen in MSs or MFPs packages differ between them, with MFPs allowing for a more uniform dehydration of the spermatozoa and a higher cryosurvival, monitored by computer assisted sperm analysis (CASA) as proportion of linearly motile spermatozoa, compared to semen packaged and processed in MSs. The organization and relative surface of biological material (veins; e.g., frozen extender, bound water, solutes and spermatozoa) as well as free water (lakes) was measured as the degree of dehydration of the samples. The apparent organization of lakes and veins differed between packages, with the MFPs depicting larger lakes than the MSs. The sizes of the lakes in the latter appeared, moreover, highly asymmetrical depending on their position of the section. The relative surface of these lakes per section, respectively veins differed between packages (P<0.05), indicating a larger amount of free-water (lakes; 81.73+/-2.07% vs. 77.91+/-1.57%) in the MFPs and, consequently, thinner veins than in MSs. In conclusion, MFPs seem to allow for a more homogenous dehydration of the spermatozoa/frozen extender compared to MSs, which might account for their somewhat better sperm quality post-thaw.
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    ABSTRACT: The present experiments were designed to study the effect of adding the detergent Equex-STM to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN(2)) vapour approximately 3 cm above the level of LN(2) for 20 min and then were plunged into LN(2). Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (p > 0.05). The results of these investigations suggested that Equex-STM exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.
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