Differences in semen freezability and intracellular ATP content between the rooster (Gallus gallus domesticus) and the Barbary partridge (Alectoris barbara)

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P < 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P < 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P < 0.0001), while ATP values significantly decreased after thawing (P < 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P < 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing.In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezabililty between the two species.     

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation.     

Semen characteristics of the captive Indian white-backed vulture (Gyps bengalensis)

The present paper describes, to our knowledge for the first time, the successful collection and evaluation of semen from the Indian white-backed vulture (Gyps bengalensis), a critically endangered bird. Over a period of 2 yr, semen was collected using the manual massage method and evaluated for semen volume, semen pH, sperm concentration, percentage normal/abnormal spermatozoa, and percentage motile spermatozoa. It appears that the concentration of spermatozoa and percentage motile spermatozoa in the Indian white-backed vultures are low compared to those in other birds. Tyrode medium supplemented with albumin, lactate, and pyruvate (TALP) proved to be the best semen extender compared to two others (Beltsville Poultry Semen Extender and Lake diluent). Furthermore, TALP with 20% egg yolk and supplemented with 8% dimethyl sulfoxide maintained 50% of the initial percentage of motile spermatozoa following cryopreservation and thawing. A computer-aided semen analysis indicated that the spermatozoa of the Indian white-backed vulture are extremely active and swim in linear trajectories for up to 5 h following dilution in TALP. The trajectories were linear with time, but we noticed a decrease in the velocity parameters (average path velocity, curvilinear velocity, and progressive velocity). Thus, the present study provides baseline data on semen characteristics of the highly endangered Indian white-backed vulture, and these data could be of immense importance to reproductive and conservation biologists attempting to breed these animals in captivity, which to date has not been achieved.     

Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders

Although glutathione content in boar spermatozoa has been previously reported, the effect of reduced glutathione (GSH) on semen parameters and the fertilizing ability of boar spermatozoa after cryopreservation has never been evaluated. In this study, GSH content was determined in ejaculated boar spermatozoa before and after cryopreservation. Semen samples were centrifuged and GSH content in the resulting pellet monitored spectrophotometrically. The fertilizing ability of frozen-thawed boar sperm was also tested in vitro by incubating sperm with in vitro matured oocytes obtained from gilts. GSH content in fresh semen was 3.84 +/- 0.21 nM GSH/10(8) sperm. Following semen cryopreservation, there was a 32% decrease in GSH content (P < 0.0001). There were significant differences in sperm GSH content between different boars and after various preservation protocols (P = 0.0102 ). The effect of addition of GSH to the freezing and thawing extenders was also evaluated. Addition of 5 mM GSH to the freezing extender did not have a significant effect on standard semen parameters or sperm fertilizing ability after thawing. In contrast, when GSH was added to the thawing extender, a dose-dependent tendency to increase in sperm fertilizing ability was observed, although no differences were observed in standard semen parameters. In summary, (i) there was a loss in GSH content after cryopreservation of boar semen; (ii) addition of GSH to the freezing extender did not result in any improvement in either standard semen parameters or sperm fertilizing ability; and (iii) addition of GSH to the thawing extender resulted in a significant increase in sperm fertilizing ability. Nevertheless, future studies must conclude if this is the case for all boars. Furthermore, since addition of GSH to the thawing extender did not result in an improvement in standard semen parameters, this suggests that during the thawing process, GSH prevents damage of a sperm property that is critical in the fertilization process but that is not measured in the routine semen analysis.     

Cryopreservation of canine semen using a coconut water extender with egg yolk and three different glycerol concentrations

Semen was collected by digital manipulation from six adult dogs. The second fraction of the ejaculate was used in this study. The semen was assessed by macroscopic and microscopic criteria 1 min after collection, first dilution, cooling, glycerol addition and thawing. Experiments were conducted to compare the effect of three different concentrations of glycerol in coconut water extender. The freezing method employed was that one described for caprine semen with slight changes. Semen was thawed at 37 degrees C for 1 min. Spermatozoal motility after thawing was 49.2+/-26, 44.2+/-18.3 and 35.8+/-26.8% for groups with 4, 6 and 8% glycerol, respectively. The vigor after thawing was 2.6+/-1.1, 2.7+/-1.0 and 2.1+/-1.2 for these groups, respectively. There was no difference among groups in motility and vigor. However, a smaller percentage of total and secondary abnormalities was observed using 6% glycerol in coconut water extender. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen using a coconut water extender.     

Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality

Human spermatozoa cryopreservation is an important means of assisted reproductive technology and male fertility preservation. Although this technique is particularly useful, sperm cryopreservation significantly reduces the quality of spermatozoa after freezing and thawing. The objective of the study is to examine the efficacy of mitochondria-targeted antioxidant MitoTEMPO in improving sperm quality during semen cryopreservation processes. Semen samples were collected and cryopreserved in extenders containing different concentrations (0.0, 0.5, 5, 50, and 500 μM) of MitoTEMPO. Sperm motility, viability, membrane integrity, mitochondrial membrane potential and antioxidant activities were measured and analyzed. The results showed that the addition of MitoTEMPO (5–50 μM) significantly improved post-thaw sperm motility, viability, membrane integrity and mitochondrial membrane potential (P < .05). Meanwhile, antioxidant enzymes activities were enhanced and MDA content were decreased in the group supplemented with MitoTEMPO. In conclusion, mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality and antioxidant enzymes profile.     

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen

The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor^®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.     

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Although the development of semen cryopreservation in the African elephants (Loxodonta africana) has been accomplished, effective procedures for cryopreservation of Asian elephant (Elephas maximus) spermatozoa have not been established. In the present study, we investigate the freezing methods for conservation of Asian elephant spermatozoa under field conditions and identify the most suitable freezing protocols which provide acceptable post-thaw semen quality. Semen was collected from two Asian elephant bulls (EM1 and EM2, 10 ejaculates from each bull) by manual manipulation and were assessed for volume, pH, sperm cell concentration, and progressive motility. Eight out of 20 ejaculates were of acceptable quality (progressive motility >/= 60%), and were used for cryopreservation studies. Semen were frozen in TEST + glycerol, TEST + DMSO, HEPT + glycerol, or HEPT + DMSO. The post-thaw progressive sperm motilities were assessed, and sperm cells were stained with PI and FITC-PNA for membrane and acrosomal integrity assessment using flow cytometry. Post-thaw progressive motility of spermatozoa (EM1: 42.0 +/- 4.3%; EM2: 26.0 +/- 17.3%) and the percentage of membrane and acrosome intact spermatozoa (EM1: 55.5 +/- 8.1%; EM2: 46.3 +/- 6.4%) cryopreserved in TEST + glycerol were significantly higher than (P < 0.05) those frozen in the other medium investigated choices for cryopreservation of Asian elephant spermatozoa. The data support the use of TEST + glycerol as an acceptable cryopreservation media of Asian elephant semen for the establishment of sperm banks.     

Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration

Quality and in vitro fertilizing ability of frozen-thawed cat semen collected by urethral catheterization (CT) or electroejaculation (EE) after medetomidine administration were compared. Sperm collection was performed by an urinary tomcat catheter and, 4 days apart, by electroejaculation from each of eight tomcats. Results showed that semen collected by CT was characterized by lower volume (10.5+/-5.3 microL, P<0.05), higher sperm concentration (1868.4+/-999.8 x 10(6)/mL, P<0.05) and lower pH (7.0+/-0.4, P<0.05) than that collected by EE (67.1+/-25.9 microL, 542.9+/-577.9 x 10(6)/mL, and 7.9+/-0.4, respectively). Spermatozoa characteristics after thawing at 0, 3 and 6h did not differ between the two methods of collection. Also cleavage rate and embryo production from oocytes fertilized with frozen-thawed spermatozoa collected by CT or EE showed no significant differences (P>0.05). In conclusion, the results obtained in the present study indicate that good quality freezable semen can be collected from cats by urethral catheterization after medetomidine administration. This new method of semen collection appears very useful in practice and, compared with the electroejaculation protocol, permits to obtain semen samples characterized by a higher concentration of spermatozoa, lower total volume and lower pH.     

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(?), Andromed(?) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(?) and Andromed(?) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(?) or Andromed(?) are used as freezing extenders.     

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.     

Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

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.     

Effect of the collection method on semen characteristics of Zebu and European type cattle in the tropics

The effect of the collection method on the characteristics of fresh semen and the recovery of spermatozoa after thawing was studied in 30 Zebu bulls (Bos indicus ) and in 30 Brown Swiss (Bos taurus ) bulls. Semen was collected by using an artificial vagina and by electroejaculation; the ejaculates were individually evaluated. Semen was diluted for freezing in skimmed milk and stored in 0.5-ml French straws, at a concentration of 30 x 10(6) spermatozoa. Data were evaluated by analysis of variance using a factorial model which included collection method, breed effect, and interaction between method and breed, with each bull as a block. Higher volume and pH of the semen was obtained following electroejaculation. Conversely, higher concentration prior to freezing and better progressive motility after thawing was observed in semen collected with an artificial vagina. No differences in motility were obtained in fresh semen between methods. Better post-thaw recovery occurred when the semen was collected by an artificial vagina, independently of the breed type.     

Prefreezing and post-thaw semen characteristics of five ram breeds collected by electroejaculation

Rams representing five breeds were electroejaculated twice weekly, during a three-week collection period. Ejaculates were evaluated for volume and concentration before freezing and for rate of motility and percentages of motile and abnormal cells both before and after freezing. Interactions between breed and collection period were evident (P<0.05) for semen volume and post-thaw values for rate of motility and percentage motile cells. Breeds differed (P<0.05) in these traits during some periods. In contrast, pre-freezing observations of rate of motility, percentage motile and abnormal cells and post-thaw percentage abnormal cells did not differ (P>0.15) among breeds. Sperm concentration per ejaculate tended to vary (P=0.11) among breeds. Semen characteristics frequently varied across collection periods. Rams within a breed differed (P<0.01) in all semen traits except post-thaw rate of motility and percentage motile cells. Semen was negatively affected by the freezing and thawing procedure. Ram within a breed and ejaculate within ram should be considered when selecting electroejaculated semen for freezing and subsequent use in artificial insemination.     

DNA fragmentation in chicken spermatozoa during cryopreservation

Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at −196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.