Post-thaw Motility Research Articles

Evaluation of different sugar extenders and large volume cryotube for sperm cryopreservation of brown‐marbled grouper ( Epinephelus fuscoguttatus )

Brown-marbled grouper (Epinephelus fuscoguttatus) is a large-bodied coral reef fish with high ecological and economic value in South-East Asia. Previous studies have developed cryopreservation protocols in 0.25- or 0.5-ml straws, but this small volume may limit the utilization of cryopreserved sperm for large-scale artificial fertilization, considering the high fecundity of brown-marbled grouper. To aid its commercial application, the main objective of the present study was to further elaborate cryopreservation protocol in this species, by evaluating the cryoprotective efficacy of different sugar extenders (glucose, sucrose or trehalose) and the feasibility of a 5-ml cryotube for cryopreservation. After frozen in a simple media containing 10% DMSO and 0.3-M sugar extender using a 0.5-ml straw, there was a deterioration in sperm motility, viability, mitochondrial membrane potential (MMP) and ATP content compared with fresh semen, but fertilization success of cryopreserved samples was not compromised at the ratio of 10,000 spermatozoa per egg. Besides, the type of sugar extender had no obvious effect on post-thaw sperm quality and fertility, thus it is suggested that glucose, sucrose or trehalose are fully interchangeable for the cryopreservation of brown-marbled grouper semen. By employing a faster freezing rate (freezing height: 2 cm vs. 3 cm) and longer thawing time (40°C, 90 s vs. 40°C, 7 s), samples cryopreserved in a 5-ml cryotube exhibited similar post-thaw motility parameters and fertility with those in 0.5-ml straw. Overall, our results demonstrate the effectiveness of new sugar extenders and the applicability of a 5-ml cryotube for sperm cryopreservation in brown-marbled grouper, which would allow for better flexibility for large-scale artificial fertilization in hatchery practice.

Effect of antioxidants in cryopreservation media on spotted halibut (Verasfer variegatus) sperm quality during cryopreservation

In fish hatcheries, sperm cryopreservation is a helpful tool for managing broodstock and artificial fertilization. However, cold shock can cause sperm injury resulting in decreasing sperm motility, low cell viability, structural damage to the plasma membrane and DNA fragmentation. This parameters are critical factor for the assessment of cryopreserved sperm for commercial scale application. In recent years, antioxidants have been increasingly useful for treating physiological issues in fish, including in reproduction and cryopreservation procedures. The purpose of this study was to examine the effects of supplementation of various antioxidants, including trehalose, reduced glutathione (GSH), Mito-TEMPO (MT), and rosmarinic acid (RA), during fish sperm cryopreservation on sperm motility, cell survival rate, and DNA damage. One-way ANOVA and Duncan's multiple range tests (p > 0.05) were used to compare the parameters. Sperm from adult fish were cryopreserved in 10% DMSO with 300 mM sucrose (1:1) supplemented with different concentrations of trehalose (0, 50, 100, 150, 200, and 250 mM), GSH (0, 2, 4, 6, 8, and 10 mM), MT (0, 25, 50, 75, 100, 125, 150, 175, and 200 μM), or RA (0, 25, 50, 75, and 100 μM). Compared with the control groups, antioxidant supplementation in sperm was more efficient in increasing post-thaw motility, maintaining cell survival rates, and inhibiting the increase in DNA damage that occurs during non-activated sperm during storage. The results demonstrated that the post-thaw motility of sperm was higher in 8 mM GSH (85.71 ± 0.04%), followed by 6 mM GSH (82.97 ± 1.30%), 75 and 25 μM RA (82.93 ± 0.93% and 82.76 ± 1.42%, respectively) and 100 mM trehalose (80.39 ± 0.61%) with not statistically different. The cell survival rate, VCL, and VSL of frozen–thawed sperm were higher in 100 mM trehalose resulted 84.22 ± 2.24%, 114.72 ± 2.00 μm/s, and 95.49 ± 0.70 μm/s, respectively. Mito-TEMPO at concentrations 100 μM resulted lower percentages of tail DNA (1.60 ± 0.08%) with not statistically different with 100 mM trehalose. In conclusion, the present study indicated that antioxidant 100 mM trehalose provided the most pronounced protective effect in improving frozen-thawed quality of frozen–thawed spotted halibut sperm.

Insights on sperm assays and cryopreservation in six Neotropical pit vipers

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane – EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.

Pre-freezing at 10 cm above liquid nitrogen surface for eight minutes resulted the best Sapera goat semen quality

Sapera goat is a dairy goat resulting from a cross between a Saanen buck and an Ettawah cross doe. These small ruminants are reared by rural households for poverty reduction and undernutrition prevention. Breeding of Sapera goat through artificial insemination (AI) technique was expected to accelerate the increase in population. Unfortunately, the frozen semen of Sapera buck is not available yet. This study aims to determine the effect of the distance between semen straw and liquid nitrogen surface during pre-freezing process on the quality of frozen semen of Sapera goats. Semen was obtained from 1.5 years old Sapera buck. The ejaculates were diluted in tris-egg yolk extender to obtain a concentration of 120 x 106 spermatozoa/mL, then equilibrated at 5°C for one hour. The extended semen was packaged in 0.5 mL French straws (60 x 106 spermatozoa/straw). The filled straws were pre-frozen in the cold handling cabinet (Minitube, Germany) held at 10, 11, 12, 13, 14, and 15 cm above liquid nitrogen respectively for T1, T2, T3, T4, T5, and T6 groups, each with four replicates. After pre-freezing for eight minutes the straws were immediately plunged, and stored in liquid nitrogen (-196°C) for two days, followed by quality assessment. The best post-thawed motility (41.75 ± 2.06 %), viability (49.00 ± 0.82 %) and morphological abnormalities (4.75 ± 0.96 %) were obtained from pre-freezing stage with a distance of 10 cm. It could be concluded that in tris-egg yolk extender, post-thawed spermatozoa viability and motility of Sapera buck were qualified for AI when fresh semen was pre-frozen at 10 cm distance of straws from the surface of liquid nitrogen for eight minutes.

Adjustment of common carp sperm concentration prior to cryopreservation: Does it matter?

Absorbance and computer-assisted sperm analysis (CASA) was used to assess the sperm concentration of common carp (Cyprinus carpio) as an alternative to hemocytometer counting. Also the effect of different sperm concentrations on post-thaw sperm parameters and fertilizing capacity were tested. The main goal of the research was to find out whether the tested methods are suitable for fast measurement of sperm concentration and if the pre-setting of sperm concentration before cryopreservation offers any advantages over dilution of sperm to a given ratio. A linear relationship of sperm concentration measured with CASA (N = 12) as well as sperm absorbance assessed with a microplate reader with spectrophotometry module (N = 18) with sperm concentration counted in a Bürker-Türk type hemocytometer was established. For cryopreservation, 4 sperm concentrations were used (0.5; 1; 2; 4 × 109 spermatozoa per ml) and compared to dilution to a ratio of 1:9. A significant positive relationship was found between the absorbance and sperm concentration (p < 0.0001, r2 = 0.8289) as well as between immotile cell concentration measured with CASA and that counted in a hemocytometer (p < 0.0001, r2 = 0.8559). No significant main effect of sperm concentration was found on any post-thaw motility parameters measured by CASA with the exception of linearity (p = 0.0056). Sperm concentration had a significant main effect (p = 0.0156) on the fertilizing capacity of sperm samples diluted to various concentrations prior to cryopreservation. Only the highest concentration of 4 × 109 spermatozoa per ml (66 ± 6% fertilization) showed a significant difference (p = 0.0121) in comparison to the pre-set dilution ratio (49 ± 5% fertilization). The results show that both spectrophotometry and CASA are suitable for the assessment of common carp sperm concentration and that maximizing sperm concentration prior to cryopreservation results in higher fertilization.

Cryopreservation of Giraffe Epidydimal Spermatozoa Using Different Extenders and Cryoprotectants.

Simple SummaryGiraffe numbers have been plummeting over the last 30 years by 30–40%. As such, efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques such as sperm cryopreservation can help preserve the genetic diversity of giraffe subspecies or enhance genetic exchange between populations. However, to date, the post-thaw motility of recovered sperm has been variable. In this study, spermatozoa were collected from the epididymides of seven giraffes to investigate whether an alternative cryoprotectant could improve sperm motility following conventional cryopreservation. For this, we compared the motility and viability of sperm prior to and after freezing in three different extenders: a commercial equine extender (BotuCrio®; Nidacon, Moedal, Schweden), a commercial bovine extender (Steridyl, Minitube, Tiefenbach, Germany), and an in-house “made” bovine egg yolk extender (TEY). Each was further supplemented with either glycerol or a mix of glycerol and methylformamide cryoprotectants. The results show that spermatozoa frozen with a mix of two cryoprotectants had significantly higher post-thaw motility compared to glycerol alone. Specifically, spermatozoa frozen in TEY and a mix of cryoprotectants achieved post-thaw sperm motility of 57 ± 3%. These results might serve as a blueprint for an improved protocol for giraffe sperm cryopreservation. Giraffe numbers have plummeted over the last 30 years by 30–40%. Thus, their conservation status has been raised from least concern to vulnerable. Efforts to manage in situ and ex situ populations are increasing. Assisted reproduction techniques (ART) such as sperm cryopreservation could help preserve the genetic diversity of giraffe subspecies and, when used for artificial inseminations, enhance genetic exchange between isolated populations. However, to date, the post-thaw motility of recovered sperm has been low and inconsistent. In this study, epididymal sperm collected from the testes of giraffes (n = 7) was frozen in three different extenders, namely, BotuCrio, Steridyl, and test egg yolk (TEY), each supplemented with one of two different cryoprotectants (5% glycerol or a mix of 1% glycerol and 4% methylformamide) and frozen over liquid nitrogen vapor. Across all three extenders, sperm showed significantly better post-thaw results when frozen with a mix of glycerol and methylformamide compared with glycerol alone. Sperm frozen with TEY and a mix of glycerol and methylformamide achieved superior post-thaw total and progressive sperm motility of 57 ± 3% and 45 ± 3%, respectively. These results show the benefit of using alternative cryoprotectants for freezing giraffe spermatozoa and could aid in the application of ARTs for giraffe subspecies or the closely related endangered Okapi.

Supplementation of melatonin to cooling and freezing extenders improves canine spermatozoa quality measures

BackgroundSperm freezing and cold storage are the two most common assisted reproductive technologies in the canine breeding industry. The freeze-thawing process causes significant detrimental changes in both sperm cell structure and function. Previous research has confirmed that excessive accumulation of un-scavenged free radicals (oxidative stress) plays an important role in the cryopreservation-induced damage to sperm cells. Also, the gradual accumulation of the free radicals during cold storage leads to a decline in the sperm quality markers. Melatonin is an endogenous neurohormone synthesized from tryptophan amino acid by pineal glands. Besides its several well-known physiologic roles, melatonin has a significant antioxidant potential through direct free radical scavenging properties. Therefore, the current study was designed to evaluate the potential in vitro protective properties of melatonin (0.5, 1, and 2 mM) on canine sperm cells after freezing or during long-term cold storage (9 days, 5 °C) on most important sperm in vitro fertility markers.ResultsMelatonin at 0.5, 1- or 2-mM concentrations could preserve significantly higher sperm total motility after 4 days of cold storage. However, only the 1- and 2 mM melatonin concentrations could result in better TM and PM values after 7 days of cold storage. Furthermore, melatonin supplementation could preserve higher sperm viability and acrosome integrity after 7 days of storage. Also, it could have significant protective effects on the cooled sperm DNA integrity. In the freezing section of the current research, melatonin at either 1- or 2-mM concentrations could not improve the sperm post-thaw TM and PM, whereas they improved sperm DNA integrity. Also, the post-thaw plasma membrane functional integrity and sperm velocity parameters were not affected by the treatment. Although DMSO (Dimethyl Sulfoxide) as the melatonin solvent could reduce the level of sperm lipid peroxidation and even improve the post-thaw sperm DNA integrity compared to the negative control, it reduced the post-thaw sperm progressive motility. However, the negative effects were reversed by concurrent melatonin supplementation at 1- and 2-mM concentrations.ConclusionThe addition of 1- or 2-mM melatonin to the canine sperm freezing and cooling media could improve sperm motility, viability, acrosome, and DNA integrity.

Semen collection, evaluation, and cryopreservation in the bonobo (Pan paniscus)

BackgroundCaptive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen.ResultsPre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40–80%), viability of 69% (38–85%) and 58% (46–72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5–25%) using the one-step freezing medium, and 19% (5–30%) using the two-step medium. Average post-thaw viability was 15% (4–24%) and 21% (15–29%), respectively.ConclusionsThis report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa.

Effect of body weight and scrotal circumference on semen production traits in murrah buffalo bulls

The study was undertaken to determine the effect of bodyweight and scrotal circumference on semen production traitsin Murrah buffalo bulls. Data on seminal characteristics,body weight, and scrotal circumference collected from 77Murrah bulls were utilized to study the effects using leastsquareanalysis under General Linear Model. Body weightand scrotal circumference showed a highly significantimpact on all semen production traits. Similarly, thecorrelation coefficients were also highly significant andpositive, except the non-correlation between scrotalcircumference and post-thaw motility. An increasing trendin seminal parameters in rise with body weight and scrotalcircumference were also observed. Furthermore, bullsweighing 540 kg and above with scrotal circumference of&gt;30 cm displayed better semen quality with higher numberof frozen semen doses per ejaculate. These findings suggestthat higher body weight and scrotal circumference couldbe considered as important selection criteria parameterswhile selecting young bulls for the purpose of frozen semenproduction to improve the frozen semen doses.

Practical Method for Freezing Buck Semen.

Simple SummaryGoat semen was previously considered to be problematic to freeze because of reactions between the semen and the components of the freezing media that were available at the time. However, there have been reports of several successful attempts to freeze goat semen in recent decades using various protocols, resulting in usable post-thaw sperm samples. In the present study, we adapted some of these methods to suit the particular conditions under which we had to work. We were able to produce thawed samples with acceptable sperm quality which were sent to a sperm bank for long-term storage.Although several protocols for cryopreserving buck semen are described in the literature, they differ widely in factors such as season and method of semen collection, extender and sperm concentration. Therefore, choosing a protocol that is suitable for a particular on-farm situation can be problematic. In the present study, semen was collected by artificial vagina from seven bucks on a farm located approximately 90 minutes’ drive away from the laboratory, about 6 weeks before the start of the goat breeding season. The semen was immediately extended in warm semen extender containing soy lecithin and was placed in an insulated box with a cold pack for up to 4 h, during semen collection from the remaining bucks and subsequent transport to the laboratory. Following centrifugation at 4 °C and resuspension in the soy lecithin extender to a sperm concentration of 800 × 106 spermatozoa/mL, 0.25 mL plastic straws were filled and frozen in racks 4 cm above the surface of liquid nitrogen. This simple protocol resulted in an acceptable post-thaw quality for all seven bucks, with a mean post-thaw motility of 55 ± 21% and mean fragmented chromatin of 3.27 ± 1.39%. Normal sperm morphology was >90% in all ejaculates. The semen was sent to a gamete bank for long-term storage.

Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility

Younger bulls typically produce lower volumes of semen per ejaculate with a lower sperm concentration than older more mature, bulls and often fail to meet semen demand using standard collection frequency schedules. The objective of this study was to assess the effect of ejaculate collection frequency on semen output, sperm quality and field fertility in young bulls under commercial conditions. Holstein Friesian bulls aged 366 ± 8 days (mean ± SEM) were assigned one of two ejaculate collection frequencies: (i) HF (n = 14 bulls), where ejaculates were collected twice a day, five days in each two-week period or (ii) LF (n = 12 bulls), where ejaculates were collected once a day, two days per week. The trial period continued until each bull reached both 20 ejaculates and 1000 marketable frozen semen straws. Subjective motility was assessed on all ejaculates pre-freeze and post-thaw (at 0 and 2 h). A subset of ejaculates were assessed post-thaw by computer-assisted sperm analysis for motility, kinematics and morphological defects and by flow cytometry for viability, membrane fluidity, acrosome integrity, reactive oxygen species and DNA fragmentation. A total of 13,846 inseminations (9,541 for HF and 4,305 for LF) were carried out on dairy cows and heifers. HF reached the 1000 straw threshold 41 days earlier than LF (P < 0.01) with the same number of ejaculates. Ejaculate volume and sperm concentration were not affected by treatment but the first ejaculate of the day (HF only) had a greater volume (P < 0.001) and sperm concentration (P < 0.05) than the second ejaculate. HF had higher pre-freeze total (P < 0.01) and gross (P < 0.05) motility than LF. HF had higher post-thaw (2 h) total and gross motility than LF (P < 0.05). Ejaculate rejection rates did not differ between treatments. There was no effect of treatment, week or ejaculate number of the day (HF only) on post-thaw motility and kinematic parameters or sperm viability, membrane fluidity, acrosome integrity and DNA fragmentation. However, HF had lower superoxide production than LF (P < 0.05). Pregnancy per artificial insemination was 64.5 ± 1.0% and 59.9 ± 1.1% for the HF and LF bulls, respectively (mean ± SEM; P = 0.05). In conclusion, collecting ejaculates more frequently from young bulls significantly reduced the number of days required to obtain 1000 straws, increased semen quality in terms of lower superoxide production and increased field fertility.

Genome-wide association study of beef bull semen attributes

BackgroundCattle production is dependent upon fertility because it results in producing offspring to offset production costs. A number of semen attributes are believed to affect fertility and are frequently measured as part of routine breeding soundness exams or semen collection procedures. The objective of this study was to perform a single-step genome-wide association study (ssGWAS) for beef bull semen attributes. Beef bull fertility phenotypes including volume (VOL), concentration (CONC), number of spermatozoa (NSP), initial motility (IMot), post-thaw motility (PTMot), three-hour post-thaw motility (3HRPTMot), percentage of normal spermatozoa (%NORM), primary abnormalities (PRIM), and secondary abnormalities (SEC) were obtained from two artificial insemination (AI) centers. A total of 1819 Angus bulls with 50,624 collection records were used for ssGWAS. A five-generation pedigree was obtained from the American Angus Association and consisted of 6521 sires and 17,136 dams. Genotypes on 1163 bulls were also obtained from the American Angus Association and utilized in ssGWAS.ResultsA multi-trait animal model was used for the estimation of single nucleotide polymorphism (SNP) effects. Significant SNP were those with a -log10P-value threshold greater than 4.0. Volume, CONC, NSP, IMot, PTMot, 3HRPTMot, %NORM, PRIM, and SEC have five, three, six, seven, two, six, six, and two genome-wide significant SNP, respectively.ConclusionsSeveral significant SNP were determined to be near or within quantitative trait loci (QTL) associated with beef bull semen attributes. In addition, genes associated with fertility were found to contain or be near the significant SNP found in the study. The results indicate there are regions of the genome that impact fertility, proving inclusion of genomic information into genetic evaluation should be advantageous for genetic improvement of male fertility traits.

Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain.

Simple SummaryEfficient collection and cryosurvival of semen from threatened wildlife species is key for the success of artificial reproductive technologies (ARTs). The viscous nature of ejaculates often collected from species such as rhinoceros, elephant, hippopotamus and primate, render the majority of spermatozoa collected useless and is therefore wasted. The enzyme papain has been used to reduce the viscosity of camelid semen but has yet to be tested in wildlife species. Therefore, the current study investigated the ability of papain to improve the yield and quality of spermatozoa collected from viscous fractions of the rhinoceros ejaculate during cryopreservation. Papain increased the quantity of useable spermatozoa collected from ejaculates, as well as the motility prior to freezing. It also improved the post-thaw motility, velocity, linearity and straightness of samples compared to spermatozoa frozen from the sperm-rich fraction of the ejaculate. There was no detrimental effect on membrane characteristics or DNA integrity. These results show that treating rhinoceros ejaculates with papain is able to salvage valuable spermatozoa and improve survival post-thaw, ultimately increasing the success of ARTs and creation of biobanks for the maintenance and survival of threatened species.The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.

Development of a Sperm Cryopreservation Protocol for Critically Endangered Mohashol, Tor tor (Hamilton).

This study dealt with the development of a sperm cryopreservation protocol of Tor tor. Sperm was collected from hormone-induced males and the concentration and pH of sperm were estimated as 4.3 ± 0.1 × 1010 cells/mL and 8.6 ± 0.2, respectively. Activation of sperm motility was evaluated in different osmolalities of NaCl solution where motility of sperm decreased with increasing osmolality of extenders, and was completely inhibited at 319 mOsmol/kg. Similarly, the swimming duration of activated sperm was affected as the osmolality of the extender increased. The duration of initial motility of sperm was recorded as 8.4 ± 0.4 minutes at 48 mOsmol/kg, while the highest motility was recorded as 68.0 ± 7.2 minutes at 128 mOsmol/kg. To evaluate the toxicity of cryoprotectants, sperm was incubated with dimethyl sulfoxide (DMSO) and methanol at 5%, 10%, and 15% concentrations, respectively, for 5-45 minutes. Alsever's solution with 5% and 10% DMSO produced better motility during 5-10 minutes of incubation and 15% DMSO seemed toxic to sperm. For the cryopreservation of sperm, Alsever's solution, egg yolk citrate, and urea egg yolk were used as extenders with DMSO and methanol. Alsever's solution with 10% DMSO provided the highest equilibration (90.0% ± 3.5%) and post-thaw (80.0% ± 3.5%) motility followed by that of 87.0% ± 2.0% and 79.0% ± 1.9% with egg yolk citrate plus DMSO, and 82.0% ± 2.6% and 78.0% ± 2.0% with urea egg yolk plus DMSO, respectively. The sperm cryopreservation protocol developed through this study can be applied for long-term preservation of genetic materials of the critically endangered T. tor, and eventually, it will be an effective tool for protecting them from extinction.

Semen Discard Rate at Fresh and Post-Thaw Stages in Indian Riverine Buffalo (Bubalus bubalis)

The quality of spermatozoa at post-thaw stage depends on many factors including the level of lipid per-oxidation (LPO) and the antioxidant profile of the ejaculates at fresh stage. Thus, the present investigation was done to evaluate discard rate of semen ejaculates at fresh and post-thaw stages in association with various semen quality parameters in Murrah buffalo. A total of 79 ejaculates from seven Murrah buffalo bulls formed part of the study. Following application of rigorous prescribed selection criteria it was observed that 17.72% (14/79) ejaculates were discarded at fresh stage due to poor quality. The initially selected ejaculates were processed for cryopreservation as per the minimum standard protocols. The study show satisfactory results for 31 (47.69%) samples for inclusion in the further AI programme (discard rate of 52.31% (34/65) at post thaw. The mean post-thaw motility (PTM; %) of the passed ejaculates was found to be 44.2±1.0. In conclusion, study results show efforts to minimize LPO and enhance total antioxidant capacity (TAC) at various processing stages shall play a vital role in reducing discard rate of the semen samples in buffalo bulls.

Clinical benefits of a modified Cryopiece system for cryopreservation of rare ejaculated and testicular spermatozoa for ICSI.

Cryopreservation of rare testicular-retrieved spermatozoa for intracytoplasmic sperm injection (ICSI) in patients with severe oligozoospermia and azoospermia remains a major challenge in clinical practice. This study evaluated the Cryopiece system as a potential technique to cryopreserve rare human spermatozoa for ICSI. Small numbers of ejaculated (24 patients) and testicular (13 patients) spermatozoa were cryopreserved using the Cryopiece system. The total number of recovered spermatozoa and motility were assessed after thawing. Thirty-seven couples underwent ICSI using spermatozoa cryopreserved by the Cryopiece system, and ICSI outcomes (rates of fertilization, embryo cleavage, and clinical pregnancy) were evaluated. The average sperm post-thaw retrieval rate was 79.1%, and motility was 29.7%. Ejaculated spermatozoa had a higher post-thaw motility (32.5%) than testicular spermatozoa (21.8%; P = 0.005). ICSI achieved a fertilization rate of 61.9%, embryo cleavage rate of 84.6%, and clinical pregnancy rate of 43.3%. The ICSI outcomes in the ejaculated and testicular frozen-thawed spermatozoa were similar. Assisted oocyte activation (AOA) after ICSI with motile (72.1%) or immotile (71.9%) spermatozoa resulted in a significantly higher fertilization rate than that when using motile spermatozoa without AOA (52.0%; P = 0.005). However, AOA did not enhance the clinical pregnancy rate (55.6% or 40.0% vs 35.3%; P = 0.703). The Cryopiece system is simple and useful for the cryopreservation of small numbers of ejaculated or testicular spermatozoa for ICSI in patients with severe oligozoospermia or nonobstructive azoospermia.

Development of an amphibian sperm biobanking protocol for genetic management and population sustainability.

Sperm cryopreservation is a vital tool in amphibian assisted reproductive technologies that aids in genetic and population management, specifically for at-risk species. Significant advancements have been made in the cryopreservation of amphibian sperm, yet there is little information on how the cryopreservation process influences fertilization and embryonic development. In this study, we tested several cryoprotective agents (CPAs) and freezing rates on sperm recovery, fertilization potential and embryo development using Fowler's toads (Anaxyrus fowleri) as a model amphibian species for application to at-risk anurans. Three cryoprotectant treatments were tested, which included 10% trehalose + 0.25% bovine serum albumin with (1) 5% N,N-dimethylformamide (DMFA); (2) 10% DMFA; or (3) 10% dimethyl sulfoxide (DMSO). Additionally, sperm in each cryoprotectant was frozen at two different rates, -32 to -45°C/min and -20 to -29°C/min. Post-thaw sperm analysis included motility, morphology, viability, fertilization success and embryo development. Results show that 10% DMFA produced significantly higher (P =0.005) post-thaw sperm motility than 5% DMFA and was similar to 10% DMSO. Furthermore, sperm frozen at -32 to -45°C/min had significantly higher post-thaw motility (P < 0.001) compared to sperm frozen at -20 to -29°C/min. We also found that embryos fertilized with sperm frozen with 5% DMFA resulted in significantly higher (P = 0.02) cleavage than 10% DMSO, yet there was no other effect of CPA on fertilization or embryo development. Furthermore, embryos fertilized with sperm frozen at -32 to -45°C/min resulted in significantly higher cleavage (P = 0.001), neurulation (P = 0.001) and hatching (P = 0.002) numbers than sperm frozen at a rate of -20 to -29°C/min. Overall, eggs fertilized with frozen-thawed sperm produced 1327 tadpoles. These results provide insight towards a biobanking strategy that can be applied to imperilled species to preserve genetic lineages and bolster offspring genetic diversity for reintroduction.

Genetic and non-genetic factors affecting conception rate of frozen semen in small holder dairy farmer system of rural India.

Fertility traits are as important as production traits in crossbred bovine population. Assessment of the fertility of bull in any frozen semen production program and evaluation of the conception rate under actual field conditions provide valuable information. The objective of this retrospective study was to estimate the effect of genetic and non-genetic influence on conception rate of frozen semen bulls maintained at Bharatiya Agro-Industries Foundation (BAIF) Pune based on the conception rate in cattle population of small holder dairy farmer system. The data comprising of 1,08,238 insemination records pertaining to 83 Holstein Friesian pure and crossbred bulls available at BAIF Pune were used to analyze conception rate. The fixed effect solutions and covariance components were estimated by linear mixed model using the restricted maximum likelihood method in WOMBAT software. The genetic correlations were estimated using bivariate analysis between post thaw motility and conception rate. The study was based on fertility related information from cows maintained in different villages of India and thus reflects the actual fertility of frozen semen used. The study was suggestive of influence of very small fraction of genetic effect and higher impact of management effect on conception rate. Fertility-related information available from this study is an invaluable asset in decision making process of breeding policies.

The effect of gonadal and extragonadal malignant cancers on sperm quality.

BackgroundMany studies have reported that testicular germ cell tumours (TGCTs) give rise to a decrease in sperm production and may further lead to infertility. However, little is known regarding sperm production in extragonadal germ cell tumours (EGCTs). We have studied the differences in the characteristics and sperm quality of patients with testicular cancer and patients with EGCTs.MethodsWe collected and analysed demographic factors, semen parameters, and the disposition of banked sperm.ResultsTGCTs accounted for the majority of malignant cancers compared with EGCTs (83.9% vs. 16.1%, respectively). There were no statistically significant differences in semen volume, pre-freeze concentration, pre-freeze motility, post-thaw concentration, post-thaw motility or recovery rate between patients with TGCTs and those with EGCTs. Both patients with TGCTs and EGCTs showed low pre-freeze and post-thaw sperm concentrations. Men with a seminoma had a higher median level of thaw-sperm motility in their ejaculate than men with non-seminomas. TGCT (67%) and EGCT (72%) patients chose to keep their specimens in storage.ConclusionsOur study provides evidence that both patients with TGCTs and EGCTs have a high risk for impaired semen quality and gonadal dysfunction. This suggests that oncologists and patients should consider the effects of both TGCTs and EGCTs on fertility among males.

Kualitas semen sapi pejantan berdasarkan umur, suhu, dan kelembaban di Taman Ternak Pendidikan Universitas Airlangga

This research was conducted at the Teaching Farm of the Faculty of Veterinary Medicine, Airlangga University, to know the effect of age, temperature, and humidity on the quality of semen of bulls which are turned into frozen semen. This research uses fresh semen samples of bulls of various ages grouped into 3 groups, young, middle age, and old groups and measures temperature and humidity during the storage process. Fresh semen is collected using an artificial vagina and then examined macroscopic and microscopic tests which is then checked for post thawing motility. Data analysis uses Analysis of Variants (ANOVA) followed by Duncan. The results showed that the percentage of spermatozoa motility of fresh semen and post thawing differed and age affected the quality of semen produced. There was no effect of temperature and humidity when processing of collecting semen. Bull’s semen at Teaching Farm Faculty of Veterinary Medicine Airlangga University has good quality and is suitable for Artificial Insemination.