Wobble Research Articles

Sperms motility of Ongole Crossbreed (OC) Bull at various age

The purpose of this study was to identify the effect of different ages on characteristics of sperms motility of Ongole Crossbreed (OC) bulls. The material used was 12 PO bulls aged 4–6 years old, distinguished into 2 age groups, namely 4-5 years old (6 heads) and 5–6 years old (6 heads). Collecting semen was done by using an electro ejaculator. Examination of motility (kinetic parameters) used SCA v. 2.1. The parameters measured include: progressive motility, motility, velocity curve linear (VCL), velocity straight linear (VSL), velocity average pathway (VAP), linearity (LIN), straightness (STR), wobble (WOB), amplitude lateral head (ALH), beat cross frequency (BCF) and hyperactivity (H) of sperms. Data were analyzed using a T-test (IBM SPSS 16). The results showed that the value of BCF in the 5–6 years old group was significantly higher (P<0.05) than the 4–5 years old group. The age of the bull affected the sperm’s kinetic parameters (BCF). The 5–6 years old group has a more regular pattern of sperms movement than the 4–5 years old group.

Effects of Apigenin and Astragalus Polysaccharide on the Cryopreservation of Bull Semen.

Simple SummaryOxidative stress in mammalian sperm can be induced during cryopreservation; therefore, it is important to inhibit oxidative stress to maintain sperm motility during cryopreservation. The present study was performed to investigate the effects of supplementing apigenin (AP), astragalus polysaccharide (APS), or their combination on an extender for bull semen freezing. Sperm viability, motility, and motion parameters; acrosome integrity and membrane integrity; endogenous antioxidant indices; and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were evaluated after semen thawing. Our results indicated that 0.2 mmol/L AP or 0.5 mg/mL APS improved the cryopreservation of bull sperm; moreover, their combination in the extender could further improve the protective effects on bull sperm post-thaw.The purpose of this study was to determine the effects of apigenin and astragalus polysaccharides on the cryopreservation of bovine semen. Apigenin, astragalus polysaccharides, or their combination were added to a frozen diluent of bovine semen. Afterwards, Computer Assisted Semen Analysis (CASA), membrane functionality, acrosome integrity, mitochondrial integrity, CAT, SOD, GSH-Px, MDA, and ROS detection were conducted. The results showed that adding 0.2 mmol/L AP or 0.5 mg/mL APS could improve the quality of frozen sperm. Compared to 0.2 mmol/L AP alone, the combination of 0.2 mmol/L AP and 0.3 mg/mL APS significantly increased the total motility (TM), average path distance (DAP), straight line distance (DSL), average path velocity (VAP), curvilinear velocity (VCL), wobble (WOB), and sperm CAT and SOD levels (p < 0.05), while reducing the ROS and MDA levels (p < 0.05). These results indicated that the addition of 0.2 mmol/L AP or 0.5 mg/mL APS alone has a protective effect on the freezing of bovine semen. Compared to the addition of 0.2 mmol/L AP, a combination of 0.2 mmol/L AP and 0.3 mg/mL APS could further improve the quality of frozen semen.

Influence of social status on growth performance, reproductive success and sperm quality of the African cichlid Aulonocara nyassae

The status of social dominance may have consequences for the well-being of cichlids. These fish show territorial and aggressive behavior and are important for behavioral studies. The aim of the present study was to evaluate sperm variables of motility, growth performance and reproductive performance according to the social status of African cichlid Aulonocara nyassae. The experiment was divided into: Phase 1 - establishment of social statuses between males, 40 males of A. nyassae were kept in four tanks (42 L useful volume) with 10 animals in each tank for 30 days. During this period, the animals were classified according to social status as dominant, subdominant and submissive fish; Phase 2 – reproduction, one male of each social status (dominant, subdominant and submissive) per tank was selected, for a total of four males for each status and a total of 12 males. One male of each social status of each of original four tanks (total 12 males) was placed in a new tank with three females, for a total of 12 new tanks, and; Phase 3 - individualization of males (removal of females), the males were separated from their females and placed individually in 12 tanks. In Phase 1, only one dominant male was detected in a group of 10 animals, between 6 and 7 subdominant animals and, 2 and 3 submissive animals. The final weight (FW) was higher for dominant fish. The sperm concentration, motility rate (MOT), Amplitude of lateral head movement (ALH), linearity (LIN), straightness (STR), wobble (WOB) and beat-cross frequency (BCF) did not show statistical difference between dominant and subdominant fish. Curvilinear velocity (VCL), Straight-line velocity (VSL) and Average path velocity (VAP) were higher for subdominant fish. Motility time was higher for the dominant male. In phase 2, the male performance was higher for dominant and subdominant than submissive males. MOT and VAP were higher for dominant males. ALH, LIN, STR, WOB and BCF were higher for subdominant males. The social status of the males did not influence the growth and reproductive variables of the females. In phase 3, the male performance was higher for dominant and subdominant fish. MOT, VCL, VSL, VAP and WOB were superior to dominant and subdominant males. LIN and STR were superior for subdominant males. BCF was superior to dominant. Therefore, sperm quality and growth performance were influenced by changes in the social statuses.

The influence of semen collection frequency on deleterious the interaction between the enzymes of the bulbourethral gland and egg yolk during the dilution of buck semen

The study aimed to evaluate the influence of semen collection frequency at different protocols on deleterious the interaction between the enzymes of the bulbourethral gland and egg yolk during the dilution and of buck semen. Five bucks aged 2.5-3 years were included in this study. Four different protocols of semen collection frequency were applied; once a week, twice a week at an interval of two days, thrice a week at an interval of one day, and twice daily at an interval of one hour. Buck semen ejaculates were evaluated using computer assisted sperm analysis that includes the percentage of motile sperm (MS), percentage of progressive motile sperm (PMS), velocity curvilinear (VCL), velocity average path (VAP), velocity straight line (VSL), linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head movement (ALH), and beat cross frequency (BCF). Results showed that sperm motility parameters had decreased significantly after dilution. Sperm motility parameters (VCL, VSL, VAP and ALH) of the second ejaculate were higher significantly (P<0.05) after dilution, when ejaculates collected twice and thrice a week and twice a day. Conversely, motility parameters of third ejaculates were less than those of first and second ejaculates when ejaculates collected thrice a week. In conclusion, there are effects of semen collection frequency on the ability of buck semen dilution, and the best results were when ejaculates had collected twice a day at an interval of one hour, accordingly we recommended this protocol for preparation of buck semen for artificial insemination.

The Response of Cockerel Semen Kinematic Parameters LIN, STR, WOB, ALH and BCF to Induced Oxidative Stress

Computer-assisted sperm class analyser (CASA) analysis of avian semen following cryopreservation indicates that their semen motility and viability parameters become compromised, due in part to oxidative stress. To mimic these observations we have treated cockerel semen with an oxidative stress inducing agent, namely hydrogen peroxide (H2O2) and monitored the motility, kinematic and viability parameters over time. Briefly, five healthy and fertile South African Venda cockerels were selected and their semen was collected using the abdominal massage technique. The semen was then treated with H2O2 at 0 &microM, 5 &microM, 50 &microM and 200 &microM concentrations for 0, 3, 16 and 24 hrs. The semen motility, kinematic and viability parameters were then determined using the CASA system while the viability was determined using the SYBR-14/PI staining. The Pearson’s correlation coefficient was determined to test the relationships between the levels of induced oxidative stress, period of exposure to oxidative stress inducing agent and the motility plus kinematic parameters. Our data revealed that in raw cockerel semen, there was high and positive correlations between total motility (TM), progressive motility (PM), rapid (RAP), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) while the kinematic parameters LIN, STR, WOB, ALH and BCF had low or negative correlations with them. Furthermore, TM, PM, RAP, VCL and VSL remained highly and positively correlated with the induced oxidative stress and also, linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) remained negatively correlated with the induced oxidative stress, after 3 hrs. After 24 hrs, TM, PM, RAP, VCL, VSL, VAP and ALH, became negatively correlated with the induced oxidative stress while LIN, STR, WOB and BCF became positively correlated with the induced oxidative stress. Conversely, when the H2O2 concentration used was correlated with motility and kinematic parameters over time, TM, PM, RAP, VCL, VSL, VAP became negatively correlated with oxidative stress while LIN, STR, WOB, ALH and BCF show negative or low correlations with the induced oxidative stress. This data indicates that LIN, STR, WOB, BCF and to some extend ALH, reveal the least correlations with the induced oxidative stress under persistent oxidative stress conditions in cockerel semen. In conclusion, cockerel semen, like buck semen, does not easily succumb to oxidative stress since the raw semen correlations of CASA analysed parameters are comparable to these observed after 3 hrs of H2O2 treatment. In addition, the oxidative stress levels tolerated by cockerel semen should not 5 &microM H2O2 oxidative stress levels. Lastly, lack of correlation between LIN, STR, WOB, BCF and ALH and induced oxidative stress can be used in cockerel semen to show intolerable cryopreservation conditions.

Comparative study of sperms motility analysis with CASA by using leja and microscope slide

The aim of this study was to compare the assessment of spermatozoa motility by using leja and microscope slide (MS). Fresh semen of four Ongole crossbred (PO) bulls were collected by using an artificial vagina. The semen with 70% sperms motility was diluted into into 30 million/ml concentration and assessed for its motility by using CASA (SCA 5.2 Microptic). The measured parameters in this study were sperms motility, progressive sperm motility, curve linear velocity (VCL), straight linear velocity (VSL), average pathway velocity (VAP), linearity (LIN), straightness (STR), wobble (WOB), hyperactivity (H), lateral head amplitude (ALH) and beat cross frequency (BCF). The data were analyzed by using independent simple t-test (IBM SPSS statistics 24). The results showed a hyperactive spermatozoa when analyzed by using leja was at 7.3%, higher than assessed with Microscope slide (2.8%). Meanwhile, other motility parameters (motility, progressive motility, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) did not show any difference. Microscope slides can be used as a means of supporting sperms motility assesment by using CASA and more efficient.

Effect of Origanum vulgare Essential Oil Supplementation on the Advanced Parameters of Mobility and on the Integrity of Human Sperm DNA.

The reduced sperm mobility is one of the most important causes of male infertility. Several reports have indicated that the treatment of subnormal sperm samples with certain agents prior to artificial insemination significantly improves the fertilizing potential of sperm. We have among others some stimulants such as pentoxifylline, relaxin, prostaglandin E, and diltiazem. In our precedent work, we had tested the effect of supplementation with three essential oils, namely, sage (Salvia officinalis), oregano (Origanum vulgare), and eucalyptus (Eucalyptus globulus), on sperm cell mobility and vitality. Oregano oil had shown interesting biological properties by giving the best values of progressive mobility and vitality. In this study, we aim to verify the effect of oregano oil supplementation on the advanced parameters of mobility and on the integrity of the sperm DNA of 25 male infertile volunteers. Our results showed that oregano oil over an incubation period of 5 to 10 min of exposure significantly improves the advanced parameters of mobility, namely, curvilinear velocity (VCL), linear velocity (VSL), the mean velocity of the path (VAP), and the amplitude of the displacement (ALH). The effect of the increase in the VCL decreased the linearity (LIN), the mean line (STR), and the mean wobble (WOB). Oregano oil at 5 min had no significant effect on the DNA fragmentation index (DFI) and sperm decondensation index (SDI). However, at 10 min, it had a significant effect on both DFI and SDI. The analysis of our results showed that this plant oil rich in terpenoids and phenolic antioxidants could be a quite good in vitro additive with high potential for the world of medically assisted reproduction.

Sorting and cryopreservation of goat sperm with or without phenolic compounds

ABSTRACT The objectives of this study were to evaluate goat sperm sorting in continuous Percoll® density gradients and gamete freezability, in the presence or absence of phenolic antioxidants. For this, semen pools were sorted, frozen, and evaluated. The non-selected group (NSg) presented lower progressive motility (PM), linearity (LIN), straightness (STR), and wobble (WOB) than the selected groups, and straight line velocity (VSL) compared to those with catechin or resveratrol. The amplitude of lateral head displacement (ALH) was higher in NSg, and quercetin reduced the mitochondrial membrane potential (MMP). After thawing, the NSg presented lower PM than the selected groups, VSL and VAP (average path velocity) than the selected group with or without catechin, LIN and WOB than the selected with or without catechin or resveratrol, and STR than the selected with catechin. Moreover, NSg presented higher ALH and BCF than the samples selected with or without catechin. Plasma membrane integrity and intact and living cells were higher in the selected groups, and MMP was lower in the NSg and the selected group with quercetin. Thus, centrifugation in Percoll® continuous density gradients is a viable methodology to select goat sperm compatible with the freezing, especially in the presence of catechin or resveratrol.

Influence of testosterone administration at the end of the breeding season on sperm cryoresistance in rams (Ovis aries) and bucks (Capra hircus)

This study examines the influence of administering testosterone at the end of the mating season, on the responses (morphometric and functional) of ram and buck sperm to freezing-thawing. Five rams were administered 25 mg testosterone propionate (TP) subcutaneously in 2 mL of olive oil twice per week (Monday and Thursday) from October 1 to 31; 5 bucks received exactly the same treatment but from November 1 to 30. Control groups were administered 2 mL of olive oil without TP twice per week over the same period. In the rams, no significant differences were seen in plasma testosterone between the treated and control groups during treatment (0.8 ± 0.2 ng/mL vs 1.5 ± 0.5 ng/mL; P > 0.05). Significant differences were seen in this respect, however, in the bucks (4.3 ± 0.8 ng/mL and 6.9 ± 0.9 ng/mL; P < 0.05). In the rams, TP treatment increased (P < 0.05) the straight-line velocity (VSL), linearity (LIN), straightness (STR) and wobble (WOB) values in fresh sperm samples. Similarly, in the frozen-thawed samples, TP treatment increased the VSL, average path velocity (VAP), LIN and WOB values (P < 0.05) compared with controls. In the bucks, treatment with TP had no effect on any measured variable in fresh sperm; frozen-thawed sperm, however, returned greater VSL, LIN, STR, and WOB values (P < 0.05) than did controls. In the rams, treatment with TP led to a reduction in all fresh sperm head morphometric variables (P < 0.05). Freezing-thawing further reduced (P < 0.05) all morphometric variables in both the control and treated groups. In the bucks, treatment with TP increased (P < 0.05) the length, area, and perimeter of fresh sperm cells, unlike that seen in ram sperm. Compared with fresh sperm, freezing-thawing led to reduced (P < 0.05) morphometric variables in both the control and treated bucks, except for the sperm head width, which in the controls remained unchanged. In conclusion, TP treatment at the end of the mating season affected fresh sperm quality, in both Spanish Merino rams and Murciano-Granadina bucks, in a species-specific manner, but improved the sperm kinetic variables after freezing-thawing in both species, apparently improving sperm cryoresistance. Treatment with TP affects the dimensions of the sperm head in a species-specific manner.

Kakule, Ayçiçeği ve Çemen Otu Tohumları Ekstraktlarının Sazan Balığı (Cyprinus carpio L.) Sperm Motilitesine ve Oksidatif Strese In Vitro Etkileri

Bu çalışmanın amacı, üç farklı bitki familyasına ait olan kakule (Trigonella foenum-graecum, Fabaceae), Ayçiçeği (Helianthus annuus, Asteraceae) ve çemen otu (Elettaria cardamomum, Zingiberaceae) tohumu ekstraktlarının in vitro koşullar altında, sazan balığı (Cyprinus carpio L.) sperm hücrelerinin motilite parametrelerine ve oksidatif stres durumlarına etkilerini belirlemektir. Bu amaçla, %0,1, %0,5, %1 ve %2 oranlarındaki tohum ekstratları ile muamele edilen sperm örnekleri 4°C‘de muhafaza edilerek, 2. ve 48. saatte ölçümleri yapılmıştır. Sperm motilite parametrelerinden kavisli hareket hızı (VCL), ortalama hızı (VAP), ileri doğru doğrusal hareket hızı (VSL), doğruluk (STR), doğrusallık (LIN), sperm başı titreşim hareketi (WOB) tayinleri bilgisayar yardımlı sperm analizi (CASA) sistemi ile gerçekleştirilmiştir. Elde edilen sonuçlara göre, Ayçiçeği ve çemen otu tohum ekstraktları, test edilen oranların tamamında motilite parametrelerini azaltıcı yönde tesir etmiştir. Buna karşın, 48. saatte %0,5 kakule tohumu ekstraktı kontrol grubu dâhil tüm gruplara kıyasla motilite parametrelerini yükseltmiştir. Ayrıca bu grupta, oksidatif stres göstergesi olarak ölçülen TBARS değerleri azalmıştır. %2 tohum ekstraktlarının tümü negatif olarak sperm örneklerini etkilemiştir. Özellikle, %2 Ayçiçeği tohum ekstratı grubu yüksek oksidatif strese neden olmuştur. Bu çalışmanın sonuçları, 4°C‘de sazan balığı sperm muhafazasında, ayçiçeği ve çemen otuna göre kakule tohum ekstraktının kullanımının daha uygun olduğunu göstermiştir.

Effects of season and single layer centrifugation on bull sperm quality in Thailand.

ObjectiveThe aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand.MethodsSemen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station;ResultsSeason had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLC-selection had a favorable effect on PRO, VAP, and WOB that differed among seasons.ConclusionOur results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

149 The second isoform of gonadotrophin-releasing hormone and its receptor affect boar semen quality

Pigs are the only livestock species encoding a functional protein for both the second isoform of gonadotrophin-releasing hormone (GnRH-II) and its cognate receptor (GnRHR-II). Unlike the classical GnRH system (GnRH-I and GnRHR-I), GnRH-II and GnRHR-II are abundantly produced in porcine testes. Moreover, GnRH-II binding its receptor on Leydig cells stimulates luteinizing hormone-independent testosterone secretion. Interestingly, GnRHR-II is also localised to the connecting piece of mature, ejaculated spermatozoa, whereas GnRH-II is detected in seminal plasma, an interaction possibly influencing the function of sperm. To examine the role of GnRH-II and its receptor in the testis, we produced a swine line with reduced endogenous GnRHR-II levels (GnRHR-II KD). The objectives of this study were to (1) compare sperm characteristics between mature GnRHR-II KD and littermate control boars on the day of collection and following semen extension and (2) determine whether a GnRHR-I and GnRHR-II antagonist alters sperm characteristics after storage of extended semen. In Experiment 1, GnRHR-II KD (n=3) and littermate control (n=3) ejaculates were collected (Day 1) and computer-assisted sperm analysis (CASA) was performed (IVOS II Animal; Hamilton Thorne) to determine measures of sperm motion (motility, progressive motility, slow, and static), morphology (normal morphology, bent tail, coiled tail, distal droplet, proximal droplet (PD), distal midpiece reflex, elongation, and area), and kinematics (length of average path (DAP), length of straight line path (DSL), length of curvilinear path (DCL), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), beat-cross frequency, and wobble (WOB)). Next, 3 billion sperm were extended with Androstar Plus (80-mL doses; Minitube) and stored at 17°C until Day 7 CASA. Data were analysed with the MIXED procedure of SAS (SAS Institute Inc.). On Day 1, semen doses from GnRHR-II KD boars had reduced DSL, VSL, STR, LIN, and WOB (P&amp;lt;0.05), whereas sperm from control boars possessed more PD (P&amp;lt;0.01). Day 7 CASA revealed that transgenic sperm had reduced DAP, DCL, VAP, and VCL, although sperm from control boars were slower (P&amp;lt;0.05). In Experiment 2, control ejaculates (n=3) were extended as above, treated with increasing concentrations (0, 0.0001, 0.001, 0.01, 0.1, 1, and 10μM) of a GnRH antagonist inhibiting both GnRHR-I and GnRHR-II (SB-75, cetrorelix), and stored at 17°C until Day 7 and 9 CASA. On Day 7, only sperm characteristics in doses treated with 10μM SB-75 were significantly lower (normal morphology, DAP, DCL, VAP, VCL, and ALH) or higher (PD, WOB, and area) than controls. Similar differences (except ALH; P&amp;lt;0.10) for the 10μM SB-75 treatment were detected on Day 9; however, motility, slow, static, STR, and LIN were also reduced (P&amp;lt;0.05). Thus, these data suggest that GnRH-II and its receptor are important to sperm function, representing a potential avenue to improve semen preservation. This research was funded by USDA/NIFA AFRI (2017-67015-26508; BRW).

Effect of three commercial extenders on sperm motility and fertility in liquid ram semen stored at 15 °C or 5 °C

The effect of different extenders on sperm motility and fertility was evaluated during liquid storage of ram semen at 5 °C and 15 °C. The semen was collected, pooled and diluted in three commercial extenders: Inra 96® (INRA) based on skimmed milk, Biladyl® A fraction (BIL) based on egg yolk, and Ovixcell® (OVIX) based on soybean lecithin. Then, sperm motility was evaluated at 0, 6, 24, 48, 72 and 96 h. In order to evaluate fertility, samples stored at 15 °C were used after dilution in INRA and OVIX. Results showed that progressive motility was significantly higher up to 72 h of storage in sperm samples maintained at 5 °C in comparison with 15 °C, similarly for each tested diluent. When samples were stored at 5 °C in OVIX, kinematic parameters such as velocity (except curvilinear velocity, VCL), trajectory [linearity (LIN), straightness (STR), wobble (WOB)], amplitude of lateral head displacement (ALH) and beat/cross frequency (BCF) were higher than in INRA and BIL. No significant differences in pregnancy rate were detected between INRA (62.6%) and OVIX (58.9%). In conclusion, liquid storage at 5 °C with OVIX extender is an interesting option since non-animal components are used, and this extender offers similar in vitro and in vivo efficacy as other extenders containing animal components.

Effects of cooling rates on the quality of Prochilodus lineatus (Valenciennes, 1836) sperm.

This study aims to investigate the effect of different cooling rates on the semen cryopreservation of curimba (Prochilodus lineatus). Nineteen ejaculates were obtained from adults males and cryopreserved at 15°C/min (CR15), 30°C/min (CR30) (controlled temperature inside and outside straw, speed was stable during freezing) and direct freezing in liquid nitrogen vapour (~35.6°C/min) (CRNV). The straws were thawed and seminal parameters evaluated. DNA fragmentation through the comet assay was assessed. A fresh sperm sample was not frozen and used for analyses. Data were submitted to an analysis of variance (ANOVA), and means were compared by Scott-Knott test (p<0.05) using the R Software. Mean motility percentage was 100%, and motility duration was 39.5±5.7s for the fresh sperm (subjective analysis); 58.9±8.0% and 24.5±5.7s for CR15; 64.8±4.8% and 26.5±7.1s for CR30; and 50.1±16% and 25.7±4.7s for CRNV, respectively. Motility percentages were higher and equal between CR15 and CR30 compared to CRNV (p<0.05). Some sperm motion kinetics, namely average path velocity (VAP) and straight line velocity (VAS), were higher for CR30 (p<0.05), while curvilinear velocity (VCL) and velocity progression (PRO) were lower for CRNV (p<0.05). Straightness (STR) and wobble (WOB) were the same among treatments (p>0.05). Sperm morphology results indicated higher means for total morphological sperm alterations in CRNV. All cooling rates caused sperm DNA fragmentation, although CR30 provided a less harmful effect. This is the first report for cryopreserved P.lineatus sperm preserved under different controlled cooling rates. The cooling rate of 30°C/min is indicated for the cryopreservation of this fish sperm as it led to the lowest detrimental spermatozoa effects.

Swimming kinematics and temperature effects on spermatozoa from wild and captive shortnose sturgeon (Acipenser brevirostrum)

Computer-assisted semen analysis (CASA) and cluster analysis were used to compare spermatozoa swimming kinematics and milt quality between wild and captive shortnose sturgeon (Acipenser brevirostrum). Milt samples from 27 shortnose sturgeon were collected in May 2016 and June 2017. Of these, 19 were wild caught in the Saint John River, New Brunswick, Canada, and eight were from a captive population at the Mactaquac Biodiversity facility. The following kinematic variables were measured immediately following sperm activation (˜5 s), at 30, 60, and 180 s post-activation; average path velocity (VAP); straight-line velocity (VSL); curvilinear velocity (VCL); amplitude of lateral head displacement (ALH); beat cross frequency (BCF); straightness (STR); linearity (LIN); wobble (WOB); percent motility (MOT). Analyses were conducted at 7, 10, and 14 °C to determine potential effects of temperature on kinematics. Principal components analysis (PCA) of original kinematic variables yielded two main components, a speed/wobble component along with a movement pattern component. Hierarchical cluster analysis (HCPC) indicated there were distinct subpopulations, with composition of clusters the result of fish source (wild-caught or captive). Wild-caught fish had greater sperm densities (P = 0.0064) and sperm swimming speeds compared to captive fish (P < 0.05). Temperature had a significant effect only on captive spermatozoa, and this result was not consistent between time periods. There was no effect of hormonal manipulation on spermatozoa motility kinematics. Results indicate there are significant differences in measures of milt quality between wild and captive shortnose sturgeon, indicating an effect of rearing condition on reproductive potential, which may affect fertilization success.

Amides as cryoprotectants for the freezing of Brycon orbignyanus sperm

The objective of this study was to evaluate the use of different amides as an alternative for the cryopreservation of sperm from the endangered species, Bryconorbignyamus, an important fish for aquaculture in Brazil. The cryoprotectants which were combined with Beltsville Thawing Solution™ extender, each at the concentrations of 2.5%, 5%, 7.5%, and 10%, were as follows: (1) dimethylacetamide (DMA), (2) dimethylformamide (DMF), and (3) methylformamide (MF). The cyoprotectant methylglycol (MG, 10%) was used as a control group. Therefore, 13 treatment groups were performed. The sperm were diluted in each cryoprotectant, loaded into 0.25-mL straws, frozen in a nitrogen vapor vessel (dry shipper), and stored in liquid nitrogen at–196 °C. The following sperm-movement parameters were measured using CASA: total motility (TM), progressive motility (PM), curvilinear speed (VCL), rectilinear speed (VSL), average path velocity (VAP), curved line distance (DCL), straight line distance (DSL), average path distance (DAP), straightness (STR), linearity (LIN), wobble (WOB), cross beat frequency (BCF) and sperm movement duration (SMD). In order to assess DNA fragmentation index, lipid peroxidation, cell integrity, integrity of plasma membrane, membrane fluidity, mitochondrial functionality, and reactive oxygen species, flow cytometry was used. The best results were obtained with 7.5% DMF, among all the amides tested, with sperm motility (66%) significantly higher than the other cryoprotectants evaluated (P < .05). DMF (7.5%) exhibited higher velocities and distances, and lower ROS production and lipoperoxidation, in comparison to MG (P < .05). It also exhibited high membrane integrity (84.5%) and cell integrity (98.7%), and low membrane fluidity (20.3%) (P < .05). MG (10%) exhibited only 38% motility, lower mitochondrial protection (39.8%), and higher DNA fragmentation index (0.07), despite the high membrane integrity observed (85.6%). The DMF concentration of 7.5% demonstrated greater protection of sperm function and cellular structure, and is therefore recommended for the cryopreservation of B. orbignyamus spermatozoa.

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using ImageJ/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.

Buck Semen Does Not Easily Succumb to Oxidative Stress

Semen processing and manipulation generally result in loss of sperm motility and sperm velocity due in part to oxidative stress. In this study we investigated the vulnerability of South African indigenous unimproved buck semen to oxidative stress induced by an oxidative stress inducing agent, namely, hydrogen peroxide (H2O2). Semen ejaculates were collected from four superior South African indigenous unimproved bucks in a total of ten collections and then each duplicate was treated with different concentrations of H2O2 in presence or absence of Dithiothreitol (DTT). Sperm motility and velocities were determined using the computer aided sperm class analyser (CASA). SYBR-14 and propidium iodide (PI) Live/Dead assay kit was used to determine cell viability and Yo-Pro-1 plus PI Apoptosis kit was used to determine apoptosis. Statistical analysis was performed on the data using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL). South African indigenous unimproved buck raw semen motility was between 97% with 98% viability and 0% apoptotic cells. Comparisons of the untreated controls at 0 and 3 hrs incubations revealed that after 3 hrs there was overall a decrease in the number viable cells with the majority of remaining cells exhibiting circular movements accompanied by high progressive (PM) and rapid (RAP) motilities. In treated South African indigenous unimproved buck semen, H2O2 marginally increased total motility (TM) with few apoptotic sperm cells while retaining high viability. Also, H2O2 increased straight line distances travelled of more than 4 fold as compared to untreated controls with no circularly moving cells. Moreover, inclusion of DTT, an antioxidant, had minimal effects on TM, RAP, curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN) and wobble (WOB) but positively affected PM, average path velocities (VAP), apoptosis and viability. Our Pearson’s correlation data revealed that only straightness (STR) was highly positively affected by H2O2. Overall, the South African indigenous unimproved buck semen resisted deterioration in TM, RAP, VCL, VAP, VSL, LIN, WOB, viability and apoptosis under oxidative stress conditions. These data suggest that the South African indigenous unimproved buck semen does not easily succumb to oxidative stress.

Semen cryopreservation in black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguins: Effects of thawing temperature on semen characteristics

In this study, there was an examination of the effect on the characteristics of cryopreserved black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguin semen, of thawing at 37 and 5 °C. For two consecutive years, semen was collected and frozen during the April-June period from six gentoo penguins, and during the October-November period from 13 black-footed penguins. After thawing, sperm motility variables were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. For the gentoo penguins, no differences were detected in the values of frozen-thawed semen characteristics after thawing at 37 or 5 °C. For the black-footed penguins, however, thawing at 5 °C resulted in greater values (P < 0.05) for straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), and wobble (WOB) as compared with thawing at 37 °C. After thawing at 37 ºC, there were greater values with gentoo penguin sperm for percentage motile sperm, progressive motility, curvilinear velocity (VCL), VSL VAP, LIN, STR, WOB and beat-cross frequency (BCF; P < 0.05) than that for black-footed penguin sperm. After thawing at 5 ºC, there were no differences in values for any variables between the two species. In conclusion, thawing temperature affects semen characteristics in a species-specific manner. The present data strongly suggest that cryopreservation procedures should be adapted for use with each penguin species. Cryopreserved black-footed penguin semen should be thawed after cryopreservation at 5 ºC, while that of gentoo penguins can be thawed at either 5 or 37 ºC.

A novel flush technique to simulate natural dispersal of spermatozoa in the female reproductive tract and expedite motility assessment of fresh ejaculated Merino (&lt;i&gt;Ovis aries&lt;/i&gt;) sperm

Motility is an important criterion in the assessment and quantification of the quality of ejaculated and epididymal sperm samples. Ovine sperm spermatozoa are particularly susceptible to cryodamage, and shortening the interval from collection to cryostorage may potentially minimize the negative effects of cryopreservation, thereby improving the post-thaw viability of ram spermatozoa. The use of the swim-up technique (SUT) in quantifying spermatozoa motility is well documented, especially for the isolation of highly motile spermatozoa for assisted reproductive purposes. However, this technique is time consuming and involves a swim-up period of 10 minutes before the motility of a sample is recorded by using computer-assisted sperm analysis (CASA) software such as the Sperm Class Analyser® (SCA®). The novel flush technique (FT) allows for capturing of sperm motility tracks via the SCA® system shortly after semen collection, that is, within a minute. This study compared fresh ejaculated sperm motility traits by using the SUT and FT. Motility evaluations were performed using 45 semen samples obtained from 15 adult Merino rams (Ovis aries) at weekly intervals. Motility recordings were captured at 100 frames per second, using a calibrated 20 μm deep Leja slide. The percentage total motile spermatozoa of samples subjected to the FT was 89.2%, which was significantly higher than that recorded by the SUT (83.9%). The results also indicated that the wobble (WOB) parameter showed significantly higher values when using the FT, and parameters curvilinear velocity (VCL) and amplitude of the lateral head displacement (ALH) indicated significantly higher values when using the SUT. Establishing the ideal spermatozoa concentration and analysis of sperm subpopulation motility characteristics would assist in the optimization of the FT, and its use in CASA motility analysis of ovine spermatozoa. Standardization of CASA analysis of ovine semen samples, which would enable the selection of quality spermatozoa samples for use in field insemination (fresh samples) or in vitro fertilization programs, and laparoscopic AI cryopreserved samples warrants further investigation.Keywords: Cryopreservation, merino, sperm class analyser, sperm motility, sperm separation technique