Supplemented stallion seminal plasma can improve impaired motility due to the dilution effect in chilled Asian elephant sperm

The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris‐glucose‐egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm.     

Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size

The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n = 693) and Norwegian Duroc (ND; n = 655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p < 0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p < 0.0001), parity (p < 0.05) and DFI (p < 0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p < 0.01). The DFI and the motility of the same semen samples were negatively correlated (p < 0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p = 0.0038 and 92.3% vs. 89.5%; p = 0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Age-related declines in ejaculate quality and sperm kinematics vary among strains of Japanese Quail (Coturnix japonica)

For successful breeding programs, it is important to quantify the useful period of a male's reproductive life and it is often done simply by measurement of semen quality. This information is lacking for Japanese quail so we tested whether there is a decline in ejaculate quality and sperm kinematics with age, and whether the decline varies among strains. Nine males (n = 9) from each of 5 strains (A, B, C, D and E) were subjected to 4 semen collections (n = 16 per male) at 8, 16, 26 and 36 weeks of age. Ejaculate volume, sperm concentration and total sperm per ejaculate were measured, and sperm kinematics were analysed using a Sperm Class Analyser (SCA^®). There was a significant effect of age for ejaculate volume, total sperm per ejaculate and per cent medium sperm. The effect of the interaction between age and strain was significant for percent progressive motile sperm, percent rapid sperm, velocity curvilinear, velocity straight line, velocity average path, linearity, straightness and beat cross frequency. Ejaculate volume peaked at Week 26 in all strains, while peak values for sperm concentration and total sperm per ejaculate were observed at Week 16 for most strains. There were declines in percent motile sperm, progressive motile sperm and rapid sperm, and in velocity curvilinear velocity, velocity straight line and velocity average path, by Week 16 for most strains. Linearity declined by Week 26 in some strains, and all strains showed a significant decline in beat cross frequency by that age. In conclusion, the ability of CASA to detect age-related changes in sperm kinematics makes it a valuable tool for identifying the best males and thus improving quail flock fertility. It is essential that breeders understand that age affects both sperm production and sperm kinematics, and that the changes vary with strain.     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Semen characteristics and biochemical composition of cloacal foam of male Japanese quails (Coturnix coturnix Japonica) fed diet incorporated with selenium

An attempt was made to investigate the effect of dietary selenium (Se) on physical and cloacal gland size, foam production, biochemical composition of foam and semen biochemical characteristics of male Japanese quail (Coturnix coturnix Japonica). Two hundred twenty‐five (225)‐day‐old male Japanese quail were randomly distributed to three dietary treatment groups for a period of 20 weeks. Each treatment comprised of three replicates, each containing 25 chicks. Three experimental diets were supplemented with 0, 0.5 and 1.0 mg Se/kg (T₁, T₂ and T_3, respectively), and diet T₁ was considered as control. Sodium selenite was used as the source of selenium. All the birds were provided with feed and water ad libitum. Cloacal foam characteristics, that is cloacal gland index and foam weight, were significantly higher in T₂ group. However, body weight, frequency of foam discharge and testes weight (left and right) did not differ significantly (p > 0.05). Physical characteristics of semen, that is semen volume and sperm concentration, did not differ (p > 0.05) among the Se‐treated groups. The sperm motility, live–dead count and abnormality improved significantly (p < 0.05) in 0.5 mg/Se‐supplemented group compared to 0 or 1.0 mg/Se‐supplemented groups. Similarly, fertility and hatchability percentages were higher (p < 0.05) in 0.5 mg/Se‐supplemented group than in control or 1.0 mg/Se‐supplemented counterparts. The biochemical characteristics of foam in terms of total protein, acid phosphatase (ACP) and nitric oxide did not differ (p > 0.05), while the concentration of glucose was higher (p < 0.05) in 0.5 mg/Se‐supplemented diet. On the other hand, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were lower (p < 0.05) in 0.5 mg/Se‐supplemented group compared to control or 1.0 mg/Se‐supplemented groups. From this study, it was concluded that supplementation of 0.5 mg Se/kg diet was beneficial for foam variables, biochemical composition of foam, semen characteristics and fertility in male Japanese quail.     

Morphological indices for canine spermatozoa based on the World Health Organization laboratory manual for human semen

Attempting to contribute to the development of a more objective morphological evaluation of dog spermatozoa, in this study the indices of multiple sperm defects (multiple abnormalities index [MAI]; teratozoospermic index [TZI]; sperm deformity index [SDI]) were calculated following the World Health Organization (WHO) guidelines. In Experiment I, the concordance of MAI, TZI and SDI with the proportions of morphologically normal spermatozoa (MNS) was evaluated in fresh ejaculated spermatozoa (dogs = 47). In Experiment II, the potential role of indices as prognostic values was assessed in spermatozoa of different origin and treatment (fresh ejaculated: n = 6; fresh epididymal: n = 6; frozen‐thawed ejaculated: n = 6) by their correlation with different semen parameters (motility, membrane integrity and acrosome status) and with an in vitro sperm function test. Samples with different proportions of MNS showed different values of SDI, the index that better represented the decline of sperm morphology in both fresh and frozen‐thawed samples (Exp. I and II; p < 0.05). No correlations between indices and semen parameters were observed (Exp. II), but when samples were evaluated collectively, negative correlations (SDI‐motility, p = 0.01; SDI‐acrosome integrity, p = 0.002) were found. Including all the defects of each spermatozoon, SDI might be a useful index during morphological analysis and better discriminates the increase in multiple defects. A more objective morphological evaluation for dog spermatozoa was achieved by the WHO method, and in vitro tests allowed to elucidate the validity of SDI as prognostic indicator of in vitro fertilizing potential.     

Influence of different anaesthetic protocols over the sperm quality on the fresh,chilled (4°C) and frozen‐thawed epididymal sperm samples in domestic dogs

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 10⁶ spermatozoa ml^?1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine–dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.     

Extenders modify the seminal plasma ability to minimize freeze‐thaw damage on ram sperm

Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze‐thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk‐based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen‐thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin‐based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p < .05), there was no effect of the extender. SP treatment was applied after freeze‐thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p < .05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p < .05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non‐capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p < .05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.     

Comparative efficiency of novel laparoscopic and routine vaginal inseminations with cryopreserved semen in rabbits

Laparoscopic artificial insemination technique (LAI) is described to overcome reduced fertility problems in sheep artificial insemination (AI) programmes with frozen semen. Later on, this technology was modified for endangered non-domestic cats to deposit low quality or reduced number of sperm cells hardly obtained by electro-ejaculation into the oviduct. This technique by passes the complex structure of cervix and efficiently transfers the sperm cells to the point of fertilization. In recent years, rabbits are becoming popular transgenic animal models producing various therapeutic and commercial products, as well as being experimental animals for disease models. The worldwide transportation of frozen semen and re-establishment of transgenic lines using AI technology has become a common practice. Therefore, this study was designed to describe a laparoscopic intrauterine insemination technique, which might assist in conceiving the animals with limited number of sperm cells. The female rabbits were laparoscopically (n = 22) or vaginally (n = 13) inseminated with frozen–thawed semen samples containing approximately 10 × 10⁶ motile sperm. The laparoscopic insemination technique provided higher pregnancy rate (45.5%) than vaginal insemination technique (7.7%) (p < .05). In conclusion, the described laparoscopic AI might be a new alternative technique, thus enabling limited or low-quality frozen sperm samples to establish pregnancy in rabbits.     

Application of the Sperm Mobility Assay to Primary Broiler Breeder Stock

A new quantitative trait affecting male fertility was discovered in the 1990s. The term sperm mobility denotes the net movement of a sperm cell population against resistance at body temperature. Even though sperm cells are self-propelled DNA delivery vehicles, their self-propulsive nature is neither uniform among sperm within an ejaculate nor among males within flocks. Such variation is evident when semen quality is evaluated by sperm penetration of an Accudenz solution, hence the term sperm mobility assay. It is noteworthy that populations showing modest variation with respect to semen volume or sperm concentration often show considerable variation with respect to sperm mobility. Likewise, it is noteworthy that broiler breeder fertility is a function of sperm mobility phenotype when hens are inseminated artificially. This article outlines the following: 1) a series of experiments in which the sensitivity of the commercial sperm mobility assay was improved using semen donors from lines of chickens selected for either low or high sperm mobility and 2) the application of the improved technique to pedigree broiler breeders. Male fertility is subject to genetic selection when sperm mobility is the selection criterion. Therefore, sperm mobility may be a useful trait for improving broiler breeder reproductive efficiency.     

Impact of cage stocking density on egg laying characteristics and related stress and immunity parameters of Japanese quails in subtropics

The aim of this study was to investigate the effects of different cage stocking densities on egg production parameters, as well as related stress and immunity indices in Japanese quails under subtropical Egyptian conditions. Two hundred and sixteen birds of Japanese quail at 14th week of age were used in this experiment. The birds were divided randomly into three groups: 60, 72 and 84. Each group subdivided into 4 replicates, where the cages' floor spaces were 200 (S₁), 167 (S₂) and 143 (S₃) cm²/bird, respectively. Birds housed at 200 cm²/bird (S₁) had superior fertility (fertility % (p = 0.013) and hatchability % (p = 0.041)), egg production (egg weight (p = 0.034) and egg mass (p = 0.001)) and immunity parameters (higher geometric mean of antibody titres against Newcastle disease virus, p = 0.024). Furthermore, they had higher internal egg quality score: albumen height (p = 0.003), yolk height (p = 0.023), yolk index (p = 0.006) and Haugh unit (p = 0.035). Birds housed at 143 cm²/bird (S₃) had the lowest total leucocytic count and lymphocyte % (p = 0.022), but the highest H/L ratio (p = 0.001). Corticosterone concentration was lower in S₁ group (p = 0.031) than that in groups housed at higher densities. Japanese quail housed at high densities revealed drop in fertility, hatchability, production and immunity parameters, indicating a detrimental effects on both welfare and economic income.     

An approach to rescue the fertility of stallions with a high level of hemospermia

A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control—pure raw semen, (b) WB50—50% (v/v) whole blood added into semen, (c) E1—WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2—WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O₂) production were immediately evaluated. Four cycles of 20 mares were randomly assigned to the experimental groups. Mares were bred with an insemination dose of 1 billion total sperm and pregnancy was diagnosed 14 days after ovulation. Sperm kinetics could not be evaluated in the WB50 samples. Total motility was lower (p < .05) in E1 than in CT and E2 samples. Progressive motility decreased (p < .05) with an increase in the percentage of blood in the samples. The PMI and PER did not differ between groups (p > .05); however, O₂ production was higher (p < .05) in WB50 than in E2 samples, while the values were intermediate (p > .05) for CT and E1 samples. The control (90%) and E2 (90%) groups had superior (p < .05) fertility than the others (WB50—0% and E1—25%). It was concluded that sperm motility and fertility of semen with a large amount of blood can be rescued by dilution with a 2:1 extender:semen ratio using a milk-based extender.     

Implications of ram sperm rheotaxis analysed by microfluidics for fertility

Rheotaxis of sperm using a microfluidic device was explored in human, mice and bull. However, the rheotaxis of ram sperm and its role in fertility are unknown. Herein, we described the sperm rheotaxis in ram using microfluidic devices and focused on rheotaxis as potential markers of in vivo fertility. Computer-assisted sperm analysis (CASA) with controlled flow velocity was used to explore the kinematic parameters of sperm, total motility and positive rheotaxis (PR). The percentage of PR was defined as the number of PR sperm cells over the number of motile sperm cells. Then, according to the percentage of PR sperm, rams were classified into two groups; sperm with ≥40% PR and <40% PR, although the two ram groups showed similar total motility and kinematic values of sperm evaluated by CASA (p > .05). Two groups of rams mated one hundred thirty ewes naturally (10 ewes/ram). In the results, the pregnancy rate was higher in ≥40% PR (94.4%) than in <40% PR (42.5%, p < .05) after natural mating. Besides, the pregnancy loss was higher in <40% PR (33.3%) than in >40% PR group (8.1%, p < .05). In conclusion, the PR examination in semen can contribute to evaluate the reproductive performance of ram that will provide valuable insights into the semen evaluation.     

Birth of Kids After Artificial Insemination with Sex‐Sorted,Frozen‐Thawed Goat Spermatozoa

Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Antioxidant supplementation,effect on post‐thaw spermatozoan function in three sturgeon species

High levels of reactive oxygen species (ROS), which may be associated with reduced sperm quality, can be detected during cryopreservation of sperm of some species. Our objective was to investigate whether the addition of antioxidants to cryopreservation extenders influenced post‐thaw sperm characteristics and fertility in Acipenser dabryanus, Acipenser sinensis and Acipenser baerii. Prior to freezing, sperm samples were diluted with a base extender as control or in extender supplemented with catalase (CAT), glutathione (GSH), cysteine (NAC), ascorbic acid (VC) or their paired combinations. Protective concentrations of CAT, GSH and VC in the three species were 25 U/ml, 0.25‐0.5 mg/ml and 0.5 mg/ml, respectively. Cysteine showed no protective effect against ROS. The addition of CAT, GSH and VC positively affected either acrosome or membrane integrity of post‐thawed sperm in the three species, as well as spermatozoan motility in A. sinensis. The combination of antioxidants did not show a positive synergistic effect. This study suggested that the use of antioxidants in the cryopreservation of sturgeon sperm has potential to decrease intracellular ROS, and consequently preserve acrosome and membrane integrity, as well as spermatozoan motility.     

Effects of supplemental conjugated linoleic acids (CLA) on fresh and post‐thaw sperm quality of Holstein bulls

This study was designed to investigate the effects of feeding‐protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top‐dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post‐thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer‐assisted), viability (Eosin–Nigrosin), membrane integrity (hypo‐osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA‐fed group compared to control group. Also, in CLA‐fed group, the proportion of post‐thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA‐fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen‐thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post‐thaw sperm quality of Holstein bulls.     

Effect of pasteurized egg yolk on the quality of cryopreserved boar semen

Egg yolk (EY, control) is an essential ingredient of diluents for boar semen cryopreservation. Pasteurized egg yolk (PEY) reduces hygienic risks in processing and is easier to standardize. The aim of this study was to evaluate the in vitro effect of PEY (treatment) on frozen-thawed boar semen. In a split-sample approach (n = 13 boars), it could be shown that there is neither an influence (p > .05) on post-thawing motility (PTM: 5, 30 and 120 min) nor on morphologically intact sperm, percentage of acrosome defects and membrane fluidity using a PEY extender compared to the control. Mitochondrial activity (p = .043), membrane integrity (p = .015) and PTM 300 min (p = .023) were slightly affected in the treatment group. Overall, sperm quality was at a high level in both experimental groups. Further studies are needed to determine the impact of PEY on the fertilizing capacity of boar ejaculates.     

Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.