In vitro evaluation of the quality and fertilizing capacity of boar semen frozen in 0.25 ml straws
ABSTRACT 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: The objective of this study was to evaluate the effect of different cryo-protectants (glycerol, dimethylacetamide and dimethylformamide alone or combined and added to lactose–egg yolk extender) on the viability of frozen/thawed semen from the Piau breed as assessed by in vitro testing. Frozen semen samples (n = 20) were used from five male swine. Five different freezing extenders, including 2% glycerol (Group 1 – G), 2% glycerol and 3% dimethylacetamide (Group 2 – GA), 2% glycerol and 3% dimethylformamide (Group 3 – GF), 5% dimethylacetamide (Group 4 – A) and 5% dimethylformamide (group 5 – F), were evaluated. To assess post-thawing sperm quality, sperm motility and morphology were evaluated. Sperm viability was determined using the hypoosmotic swelling test, supravital staining, and a fluorescent assay (carboxyfluorescein diacetate and propidium iodide). The mean total sperm motility of semen immediately after thawing was 46.2 ± 1.3, 57.7 ± 1.5, 53.2 ± 2.1, 51.7 ± 1.2, and 46.5 ± 1.6% for groups 1–5, respectively. Groups 2 (GA) and 3 (GF) had greater motility values (P < 0.05). Fluorescent assay values of 22.3 ± 2.3%, 35.2 ± 3.7%, 30.8 ± 3.4%, 36.6 ± 3.7%, and 26.5 ± 3.8% were obtained for Groups 1–5, respectively, showing that Group 4 (A) sperm had greater viability than those from Group 1 (G), although there was no differences between the other treatments (P > 0.05). The other complementary tests (hypoosmotic swelling test and supra-vital staining) demonstrated that sperm in Groups 2 (GA), 3 (GF) and 4 (A) had the greatest viability and there were no significant differences among these three groups (P > 0.05). The most effective cryo-protectant combinations likely minimized and controlled the deleterious processes that occur in the sperm cell during freezing/thawing, thus improving post-thawing sperm viability. In conclusion, the combination of amides (3%) and glycerol (2%) or dimethylacetamide (5%) alone were more efficient at cryo-protection than glycerol alone for semen freezing in the Piau swine breed.Animal reproduction science 05/2014; 146(3-4). DOI:10.1016/j.anireprosci.2014.02.018 · 1.58 Impact Factor
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ABSTRACT: The aim of the present study was to evaluate the effect of the holding time at 15 °C prior to cryopreservation (2, 4 and 8 hours), thawing rate (37 °C for 20 seconds or 70 °C for 8 seconds) and post-thaw incubation temperature (15 °C or 37 °C) on the post-thaw boar sperm quality. These are important time periods in the freezing-thawing process which have been less studied. Sperm-rich ejaculate fractions from three healthy boars were collected once a week for five consecutive weeks and were cryopreserved with the lactose-egg yolk extender (LEY). Sperm quality was determined by assessing the motility, the acrosome status, and the sperm plasma membrane integrity at 30, 150 and 240 minutes of incubation. The results show that with the holding time at 15 °C prior to cryopreservation there was not a clear effect until at least 24 hours of holding time. The thawing rate and the post-thaw incubation temperature, however, had a marked effect on sperm quality. When the samples were thawed at 70 °C for 8 seconds, the sperm viability, motility and some kinetic variables (VCL, VSL, VAP and ALH) were greater than with results observed when the samples were thawed at 37 °C for 20 seconds. In addition after thawing the sperm samples incubated at 15 °C had a sustained sperm quality for longer, up to 4 hours post-thawing.Animal reproduction science 01/2013; DOI:10.1016/j.anireprosci.2013.12.011 · 1.58 Impact Factor
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ABSTRACT: Frozen-thawed boar sperm (FTS) has reduced motility and viability compared to cooled semen. Motility of FTS is related to in-vitro and in-vivo fertility, but this effect has not been determined in relation to the timing of ovulation. To test the effect of variable FTS motility in a multiple AI system, ejaculates from 38 boars were collected and frozen in 0.5 mL straws. Upon thawing, samples were classified (mean ± SEM) by motility as Poor (P, 20.2 ± 1.1%), Moderate (M, 31.3 ± 0.9%), or Good (G, 43.5 ± 0.8%). In replicates, mature gilts were synchronized and checked for estrus at 12-h intervals and assigned (n = 207) to receive 4.0 billion total sperm in each AI at 24 and 36-h after onset of estrus using the treatments: 1) P and M (P-M); 2) M and P (M-P); 3) G and M (G-M); and 4) M and G (M-G). For each treatment combination, a set of three boars were randomly selected within motility class for their allelic distinction with M sperm from a single boar represented across all treatments and sires used in both 1st and 2nd inseminations. The insemination to ovulation interval (IOI) was determined using ultrasound every 12-h. Reproductive tracts were collected at ∼ d-32 after AI. Treatment did not interact with IOI (P > 0.10) and did not affect (P > 0.10) pregnancy rate (57, 67, 71, 76 ± 7.2%, pooled SEM) or total number of fetuses (9.2, 9.1, 9.5, 10.0 ± 0.8) for P-M, M-P, G-M, and M-G treatments, respectively. Treatment did affect (P < 0.05) the number of fetuses sired from the 1st AI (3.1, 7.2, 6.4, 6.3 ± 1.2) and 2nd AI (5.7, 2.6, 3.0, 3.6 ± 0.9) for the P-M, M-P, G-M, and M-G treatments, respectively. The IOI also influenced (P < 0.05) the proportion of offspring sired by the 2nd AI (30.0, 57.7, 51.3, 18.3, ± 6.5%), as well as the number of fetuses sired by each AI. These results indicate FTS motility had no effect on pregnancy rate or litter size, but did affect the number of fetuses sired from the 1st and 2nd inseminations. The 1st AI appears to sire most of the litter except when P sperm was used. Number of fetuses sired was reduced when P sperm was used in either insemination compared to M, although no difference was evident between M and G. Fetal paternity appears to be a more sensitive marker for identifying the effects of sperm quality and IOI in a multiple AI system with use of FTS. These results suggest that use of semen of various quality can be used in combinations to aid in pregnancy establishment and contribute to litter size.Journal of Animal Science 10/2013; 91(12). DOI:10.2527/jas.2013-6867 · 1.92 Impact Factor