Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

This study was designed to compare commercially available extender Bioxcell^® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 10⁶ spermatozoa/ml) in tris-citric egg yolk or Bioxcell^® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell^® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell^® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell^® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.     

Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo bulls (Bubalus bubalis) through supplementation of fat

The aim of the study was to investigate the effects of dietary fat on quality of liquid and frozen-thawed semen of Nili-Ravi buffalo bulls. Adult bulls (n = 21) were fed a balanced ration (Con; n = 7) or the same ration either containing sunflower oil (SF-O; n = 7) or whole sunflower seeds (SF-S; n = 7) for 63 days. Body weight and body condition score of each bull was recorded on days 0, 30 and 60 of the experiment. Semen was collected on days 39, 46, 53 and 60, frozen by a fast method and stored at −196 °C for 24 h. Sperm motility was assessed using a bright field microscope. Plasma membrane integrity of fresh and frozen-thawed spermatozoa was assessed using a hypo-osmotic swelling (HOS) assay. The concentration of spermatozoa and volume of semen was not different among groups on various days of collection. Sunflower-enriched diets did not affect the motility and number of HOS-positive spermatozoa in the fresh semen. Motility and HOS of post-thawed spermatozoa were higher (p < 0.05) in bulls fed the sunflower-enriched diets. Similarly, diets did not affect the body condition score and body weight of bulls. In conclusion, feeding of sunflower oil or sunflower seed as fat sources can improve the quality of buffalo bull spermatozoa.     

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility,plasmalemma, mitochondrial membrane potential,acrosome, and DNA integrities,and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 μ M l-tryptophan, D2 = 50 μ M l-tryptophan, D3 = 75 μ M l-tryptophan, and D4 = 100 μ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.     

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.     

Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed

The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 × 10⁶ spermatozoa/straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at −152 °C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in an ultra-low freezer at −152 °C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher (p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen.     

Freezing of swamp buffalo semen

The French mini straw technique (Society I.M.V.) was used to preserve semen of Indonesian swamp buffalo (Bubalus bubalis) in a lactose based extender with and without the removal of seminal plasma. Extended semen with 60–70% motility before freezing showed 60 and 40–50% motility after thawing at 4°C for 5 and 180 min. respectively. Nine of 13 cows conceived to a single insemination with frozen semen. Neither post-thawing motility nor conception was improved by removing seminal plasma before freezing     

Semen cryopreservation in the Indian rhinoceros (Rhinoceros unicornis)

The objective was to identify an extender and cryoprotectant combination for Indian rhinoceros (Rhinoceros unicornis) sperm that yielded high post-thaw sperm quality. Male Indian rhinoceroses (n = 6; 7.5-34 yr old) were anesthetized and subjected to a regimented electroejaculation procedure (75-100 mAmps; 4-10 volts; 7-150 stimuli; total of 10 electroejaculation procedures). High quality semen fractions from each ejaculate were divided into four aliquots and a 2 x 2 factorial design used to compare the effect of two sperm extenders (standard equine [EQ] and skim milk-egg-yolk-sugar [SMEY]), and two cryoprotectants (glycerol and dimethylsulfoxide [DMSO]). Cyropreserved samples were thawed and assessed for motility, viability and acrosome integrity over time. Electroejaculate fractions processed for cryopreservation had high sperm concentration (516 × 10⁶/mL) and motility (79%). Post-thaw sperm characteristics were higher (P < 0.05) when semen was cryopreserved in EQ versus SMEY. Post-thaw motility of sperm cyropreserved in EQ averaged 50-55% compared to 22-37% in SMEY, with no significant differences in sperm characteristics of samples cyropreserved in glycerol and DMSO. In conclusion, sperm collected from Indian rhinoceroses via electroejaculation were cryopreserved using EQ extender with either glycerol or DMSO; post-thaw quality was adequate for use in assisted reproductive procedures.     

Impact of in vitro fertilization by refrigerated versus frozen buffalo semen on developmental competence of buffalo embryos

The objective of this study was to evaluate the fertility of buffalo semen for in vitro embryo production (IVEP) by comparing the effectiveness of refrigerated versus frozen semen. Three OPU sessions were held at 30-day intervals. For oocyte fertilization three buffalo bulls were used, one per session. At each OPU-IVEP session, one ejaculate was collected and divided into two equal aliquots. Each aliquot was either refrigerated at 5ºC/24 hours or frozen. A TRIS extender containing 10% low density lipoproteins, 0.5% lecithin and 10 mM acetylcysteine was used adding 7% glycerol for freezing. Sperm motility/kinetic was evaluated by CASA and sperm membrane integrity by the hypoosmotic swelling test. The evaluations were performed at 0 h (post final dilution at 37ºC), at 4 and 24 hs post-incubation at 5ºC and post-thaw. At 24 hs incubation and immediately post thaw sperm cells were used for in vitro fertilization of buffalo oocytes equally distributed between both groups. Cleavage rates and embryo development were followed. The embryo/matured and embryo/cultured rates were 25.4 x 14.0% and 29.4 x 18.5% (P<0.05), for chilled and frozen semen, respectively. It is concluded that cooled semen can be used for in vitro embryo production in buffalo and that a better efficiency may be expected for cooled compared to frozen semen.     

Transaminases in cattle and buffalo (Bubalus bubalis) semen in relation to fertility and seminal characteristics during moderate and colder seasons

Semen from eight (four cattle and four buffalo) bulls was collected and analysed for physical characteristics and transaminase activity. The values of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) activity were 1567±63 and 368±25 SF units¹ in the whole semen and 807±31 and 121±11 SF units in the seminal plasma for cattle and 1359±48 and 279±29 in the whole semen and 635±28 and 94±11 in the seminal plasma for buffalo. Cattle semen had significantly (P<0.01) higher mass motility, sperm concentration and transaminase activity than did buffalo semen. The enzyme activity was positively correlated with mass motility, sperm concentration and fertility, while only GOT activity was inversely related to the ambient temperature.     

Comparison of conventional and computer-assisted semen analysis in cockatiels (Nymphicus hollandicus) and evaluation of different insemination dosages for artificial insemination

Many psittacine species are threatened in the wild and also rare in captivity. Therefore, successful conservation and breeding programs are important to save these species. Unfortunately, clutches in conservation programs are frequently infertile. Semen evaluation is beneficial to investigate the causes of infertility and is advisable before artificial insemination (AI). In this study, we analyzed the semen of cockatiels (Nymphicus hollandicus) using two different methods and investigated different insemination dosages for AI. Cockatiels (n = 30) were divided into two groups (group A: nine males; group B: six males). The males in group B were endoscopically sterilized, whereas the males in group A were used as semen donors. In the first part of the study, the semen of males in group A was evaluated by semen analysis. Semen samples were collected by the massage technique and examined using a conventional light microscope and a computer-assisted semen analyzer for comparison. Results demonstrated that the evaluations of motility, progressive motility, and sperm concentration, but not of live/dead ratio, correlated strongly for both methods. However, the results for sperm concentration, progressive motility, and live/dead ratio differed significantly. In the second part of our study, the volume and quantity of spermatozoa of the semen samples were adjusted and used for AI of females of group B. Intravaginal insemination with 250,000 spermatozoa resulted in five of 17 (29%) eggs fertilized; however, intracloacal insemination resulted in only four of 57 (7%) eggs fertilized at 232,000 and 250,000 spermatozoa but none at higher or lower dosages.     

The Effect of Low-Level Laser Irradiation on Sperm Motility,and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm

Background and Objective

Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm.

Study Design/Materials and Methods

We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS - Integrated Semen Analysis System.

Results

Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05).

Conclusion

We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was more effective than a dose of 6 joules in preserving sperm motility.     

Comparison of two different cryopreservation protocols for freezing goat semen

In this study, two different semen cryopreservation protocols were compared to freeze goat semen. The ejaculates (n = 12) were collected by using electro-ejaculator from six mature bucks (two ejaculates per each buck). Each ejaculate was divided into two groups as Protocol 1 (P1) and Protocol 2 (P2). In P1, semen was diluted directly in an extender containing 15% egg yolk, 300 mM Tris, 28 mM glucose, 95 mM citric acid 5% glycerol to a concentration of 200 × 10⁶ sperm/mL. In P2, after the removal of seminal plasma by centrifugation, the semen sample was diluted with the first portion of milk extender consist of 100 mg/mL skimmed milk powder and 27.75 mM glucose (without glycerol) to a concentration of 400 × 10⁶ sperm/mL. The second portion of the milk extender containing 14% glycerol was added to semen gradually in order to achieve sperm concentration 200 × 10⁶ sperm/mL and 7% glycerol level in the final volume. Extended semen was loaded in 0.25 mL straws, held for 2 h at 4 °C, frozen in nitrogen vapor and stored in liquid nitrogen. Post-thaw motility and live sperm rate (mean ± SEM) were significantly lower (P < 0.05) in P1 as compared to P2 (47.50 ± 1.23% vs. 55.63 ± 1.72%; 80.04 ± 1.29% vs. 84.04 ± 1.08%, respectively). However, live intact, total intact, abnormal, reacted acrosome and DNA damaged sperm rates were similar (P > 0.05) in both protocols. It was concluded that both protocols used in this study provided reasonable post-thaw parameters; however, P2 yielded better motility and live sperm rate compared to P1.     

Successful preservation of capercaillie (Tetrao urogallus L.) semen in liquid and frozen states

Experiments on semen collection and preservation were undertaken by Wroc?aw University of Environmental and Life Sciences and Forestry Wis?a, Poland to assist in the protection of the capercaillie (Tetrao urogallus L.) and to create an ex situ in vitro cryobank. Semen was collected from 11 captive-bred males, using dorsoabdominal massage. Ejaculates once obtained were diluted 3-fold at room temperature with EK diluent and then a number of them were stored at 4 °C for 18, 24, and 48 hours, while the remaining ejaculates were equilibrated with 6% dimethylacetamide and frozen by pipetting, drop-by-drop directly onto a liquid nitrogen surface. Frozen pellets were thawed at 60 °C in a water bath after 4 to 28 mo of storage. In total, 103 individually collected ejaculates (54 stored as liquid and 49 frozen in liquid nitrogen) were of appropriate value for further processing. The volume of ejaculates varied from 30 to 240 μL; spermatozoa concentration from 70 × 10⁶ mL⁻¹ to 1950 × 10⁶ mL⁻¹. The total amount of live spermatozoa in the fresh semen varied from 85.3% to 99.0%, of which from 41.1% to 85.3% were morphologically normal. Among morphologically abnormal forms, bulb-head (5.6% to 36.0%) and midpiece deformations (1.3% to 16.6%) were the most frequent. Dilution and semen storage up to 24 h at 4 °C did not affect the semen quality, as far as motility and sperm morphology are concerned. A significant (P < 0.05) decrease in total live (94.9 vs. 91.7%) and live normal cells (66.4 vs. 56.7%) was observed after 48 h. About 30% to 40% of spermatozoa remained motile. Cryopreservation significantly decreased (P < 0.05) the total number of live and live normal spermatozoa however, in relation to the fresh semen, their average content was 44.1% and 37.4%, respectively. Significant (P < 0.05) individual differences were observed in the quality of the fresh, liquid stored and the frozen-thawed semen assessed in terms of spermatozoa motility and morphology. After a single insemination with thawed semen containing 9.7 million live normal cells, 80% fertility and 100% hatchability were achieved. The obtained results indicate for the first time that there is the potential to use liquid stored and cryopreserved capercaillie semen to support conservation measures for the maintenance of genetic diversity, as well as to increase the number of reintroduced progeny of this endangered grouse species.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR)

In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility.Samples of stallion semen (n = 390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility of stallions whose sperm did not contain viral DNA, were compared using the Student's t-test.EHV-4 viral DNA was not detected in any of the semen samples. EHV-1 DNA was identified in 51 of the 390 samples, (13%). One hundred and eighty-two samples came from 6 studs and there was significant difference (p < 0.05) among the proportion of stallions whose semen tested positive for viral DNA from 0 to 55% between the studs.There was a significant difference (p < 0.014) between the fertility of stallions whose semen tested positive for viral DNA and those whose semen was free from viral DNA. The stallions that excreted the EHV-1 virus in their semen appeared to be more fertile than the non-excretors, but this difference was in fact related to the breeding technique since higher proportion of excretors were found among those whose semen was used fresh rather than preserved by cooling or freezing.In conclusion, this study suggests that the EHV-1 virus may be transmitted via the semen at mating or by artificial insemination as demonstrated with other herpes viruses in other species.     

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.     

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: Comparison to Triladyl and Bioxcell

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl^®, Bioxcell^®) and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurus × Bos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 × 10⁶, 60 × 10⁶ and 20 × 10⁶ sperm/mL, corresponding to 30 × 10⁶, 15 × 10⁶ and 5 × 10⁶ sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell^® and Triladyl^® (p < 0.05), but no significant difference was observed between Triladyl^® and Bioxcell^®. Therefore we can conclude that LDL extender could be used instead of Triladyl^® or Bioxcell^® at low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.     

Improvement of stored turkey semen quality as a result of He-Ne laser irradiation

Two experiments were carried out to evaluate the effects of He-Ne laser irradiation at various energy doses on the quality of stored turkey semen. Four semen pools were used in Experiment 1. Each pool was divided into 10 aliquots, nine of which were irradiated with energy doses ranging from 0.144 to 10.8 J/cm2 while the tenth one was not irradiated (control). Each sample was evaluated for motility immediately after irradiation, 24 and 48 h later. Energy doses ranging from 3.24 to 5.4 J/cm2 had higher (P <0.01) sperm motility index (SMI) value compared to the control and samples irradiated with lower and higher laser doses. The energy dose of 3.96 J/cm2 was selected for Experiment 2 to obtain further insight on its effects on turkey sperm preservation for up to 60 h. Each pool of four semen was divided into two aliquots: one represented the control and the other one was irradiated with He-Ne laser at an energy dose of 3.96 J/cm2. Each sample was evaluated for motility and viability immediately after irradiation and then at 12 h intervals up to 60 h. The cell energy charge was also measured by HPLC. Exposure to 3.96 J/cm2 increased the SMI and viability of turkey semen stored for 60 h compared to the control (P <0.05). The cell energy charge of irradiated samples was 200% higher than in the control. Laser irradiation increased the longevity of stored turkey spermatozoa, and might be a useful technique to enhance semen quality in long-term storage.     

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.