Hypo-osmotic Swelling Test Research Articles

Effect of Addition of Zn, Cysteine, PGF2α and their Combination on Holstein Bulls Cooled Semen in vitro

The study was conducted to investigate the effect of addition of zinc sulphate, cysteine, Prostaglandin F2α (PGF2α) and their combination to the diluted semen on semen characteristics of Holstein bulls after different periods of cooling. The study was conducted on Artificial insemination Center/ Directorate of Animal Resources/ Ministry of Agriculture at Abu-Graib at the west of Baghdad during the period of Aug. 2019 to the Dec. 2019. Seven Holstein bulls were used, Aged between 2.5- 3 years. Semen was collected via Artificial Vagina one ejaculate per a week for three months. Fresh semen was evaluated which were of bad grand pooled semen were divided into 5 parts. The first part (T1) only diluted semen (Tris) serve as a control. The 2nd part (T2) added 0.576 mmol/ ml of zinc sulphate to diluted semen. The 3rd part (T3) added 5 mmol/ ml of cysteine to diluted semen. The 4th part (T4) added 37.5 pg/ ml of PGF2α to diluted semen to diluted semen. The 5th part (T5) added a combination of previous components to the diluted semen. Then the semen evaluated at 5C° after 24, 48 and 72 hrs. The results showed that the addition of zinc sulphate on cooled diluted semen have no significant difference between different periods in the percent of sperm individual motility. Addition of zinc sulphate and cysteine showed a significant increase (P<0.05) in the percent of sperm liveability. The addition of a combination (zn sulphate, cysteine and PGF2α) showed a significant decrease (P<0.05) in sperm abnormalities percent especially at 5C°. The results also showed the addition of zinc sulphate and PGF2α increase the percent of HOST (Hypo-osmotic swelling test) of spermatozoa at 48 hrs. after cooling. zinc sulphate addition showed a significant increase (P<0.05) at 5C° during as comported with other cooled period in the HOST. The addition of zinc sulphate showed a significant increase in cell membrane integrity at 5C° cooled semen. It was concluded that addition of antioxidant and hormone might improve semen quality of Holstein bulls.

Specific tail swelling pattern in hypo-osmotic solution as a predictor of DNA fragmentation status in human spermatozoa.

ObjectiveThe aim of this study was to investigate DNA fragmentation status in human spermatozoa according to specific tail swelling patterns determined via hypo-osmotic swelling test (HOST).MethodsFrozen semen samples from 21 healthy donors were thawed and prepared by the swim-up technique for use in intracytoplasmic sperm injection. The semen samples were treated for 5 minutes as part of the HOST procedure and then underwent the sperm chromatin dispersion test using a Halosperm kit. DNA fragmentation status (large halo, medium halo, small halo, no halo, or degraded) and the specific tail swelling pattern (“a”–“g”) were assessed at the level of a single spermatozoon. A total of 42,000 spermatozoa were analyzed, and the percentage of spermatozoa without DNA fragmentation (as evidenced by a large or medium halo) was assessed according to the specific tail swelling patterns observed.ResultsThe HOST examinations showed that >93% of spermatozoa across all types displayed no DNA fragmentation. The percentage of spermatozoa without DNA fragmentation was 100% in type “d”, 98.67% in type “g”, and 98.17% in type “f” spermatozoa.ConclusionWe found that the type “d” spermatozoa displayed no DNA fragmentation, but the other types of spermatozoa also displayed very low rates of DNA fragmentation. This result may be associated with the processing of the spermatozoa by density gradient centrifugation and the swim-up technique.

Effect of various antioxidants and their combinations on bull semen cryopreservation

Our objective was to evaluate the effects of adding 50 mM trehalose (TRE), 1 mM cysteine (CYS), or 400 µg catalase (CAT), individually or in combinations, to a Tris-based semen extender and determine the effects on fresh, chilled-equilibrated, and frozen-thawed bull sperm. Ejaculates from three Holstein bulls (1-3 years old) were pooled, divided into six equal portions, and put into a Tris-based extender supplemented with no additive (control), TRE, CYS, CAT, TRE + CYS, or TRE + CYS + CAT. In chilled-equilibrated semen, motility was highest in CAT but lowest in the control (P < 0.05). In thawed semen, motility was lower (P < 0.05) in CAT than in all other groups, which were not different. Based on a hypoosmotic swelling test (HOS test), TRE, CYS, and their combinations had higher (P < 0.05) percentages of sperm with an intact plasma membrane than the control or CAT groups. The TRE group had the least (P < 0.05) morphologically abnormal sperm. Plasma membrane and acrosomal integrity (PMAI) and high mitochondrial membrane potential (HMMP) values were highest in TRE and TRE + CYS and were lower (P < 0.05) in the control. Furthermore, HMMP was positively correlated with PMAI and HOS tests, but negatively correlated with major + minor abnormalities (r = 0.66, 0.242, and -0.349). In conclusion, the addition of TRE, with or without CYS, to a Tris-based semen extender improved several aspects of the frozen-thawed quality of bull semen. However, addition of CAT, by itself or in combination with other additives, did not protect bull sperm against cryopreservation-induced defects.

Idebenone improves quality of ram sperm by mitigating oxidative stress during cryopreservation

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 μM idebenone (Id2), 5 μM idebenone (Id5), 7.5 μM idebenone (Id7.5) and 10 μM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 μM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.

Sodium alginate potentiates antioxidants, cryoprotection and antibacterial activities of egg yolk extender during semen cryopreservation in buffalo

The study was conducted to determine effects of sodium alginate on sperm during cryopreservation. Each ejaculate (n = 20) of five buffalo bulls (3–5 years) were divided into six equal fractions and diluted using egg yolk based extender supplemented with different concentrations of sodium alginate and cryopreserved. Frozen-thawed semen samples were evaluated using the CASA, hypo-osmotic swelling test, cervical mucus penetration capacity test, and chlortetracycline fluorescence assay (CTC). Phosphorylation of tyrosine containing proteins and malondialdehyde concentration of sperm membrane were evaluated using immunoblotting and thiobarbituric acid reactive substance assay respectively. The semen extender’s anioxidative capacities were estimated by conducting 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays, metal chelating capacity by assessing ferrozine and antibacterial capacity using agar plate methods. Supplementation of sodium alginate in extender improved sperm longevity, plasma membrane integrity as well as capacity to transit through the cervical mucus. Supplementation of extender with sodium alginate minimises the phase transition of sperm membranes and phosphorylation of tyrosine containing proteins during cryopreservation. Malondialdehyde concentration of sperm was less in sodium alginate-treated sperm as compared with control samples. The results indicated that sodium alginate increased antioxidant capacity of semen extender. Supplementation with sodium alginate also improved the metal chelating capacity and antibacterial properties of the extender. In conclusion, supplementation of extender with sodium alginate enhances free radical scavenging, metal reduction and chelating capacities to protect sperm during cryopreservation.

Impact of Chronic Hepatitis B Virus Infection On Semen Parameters of Fertile Men

Aim: To investigate the effect of chronic hepatitis B virus (CHB) infection on semen parameters of males with proven fertility. Material &amp; methods: 61 fertile men with CHB infection and a control group of 42 HBV-negative fertile men were assessed for serum and semen HBV viral loads by quantitative real-time PCR, complete semen analysis, hypo-osmotic swelling test, seminal total peroxide, total antioxidant capacity assay and sperm DNA integrity evaluation. Results: CHB men demonstrated significantly lower sperm motility, sperm viability and sperm DNA integrity, seminal total antioxidant capacity assay and significantly increased seminal total peroxide and oxidative stress index compared with controls. CHB cases with positive semen HBV DNA showed a significant increase in seminal total peroxide and oxidative stress index compared with CHB cases with negative semen HBV DNA. Conclusion: CHB has a negative burden on seminal parameters in fertile men.

How to Increase Post-Thaw Semen Quality in Poor Freezing Stallions: Preliminary Results of the Promising Role of Seminal Plasma Added after Thawing.

Simple SummaryApproximately half of all stallions are considered poor freezers, which means that they produce ejaculate with poor cryopreservation tolerance. Although the quality of the frozen–thawed semen of poor freezing stallions is low, they can carry and transfer desirable traits to future generations via insemination. Therefore, increasing the post-thaw quality of cryopreserved semen from these stallions is an important step in efficient horse breeding. In this study, we aimed to find a way to increase the quality of cryopreserved semen from poor freezing stallions. We studied the effects of different seminal plasma (SP) additions on various functional parameters of spermatozoa. The results show that the addition of different types of seminal plasma positively affects motility and maintains the status of the plasma membrane of poor freezing stallions. Since the percentage of stallions with poor freezability is quite large, future research should target the discovery of specific seminal plasma components that could be used to increase the post-thaw quality of their spermatozoa.The aim of this study was to evaluate the effect of the addition of two types of seminal plasma (SP) after thawing on the functional characteristics of frozen–thawed (F–T) spermatozoa of poor freezing stallions during prolonged incubation periods. Seminal plasma from stallions with 35–40% (standard seminal plasma, (S-SP)) and 60–70% (above standard seminal plasma, (A-SP)) progressively motile spermatozoa after thawing was used. The motility, kinematic parameters (Computer Assisted Sperm Analysis), distribution of spermatozoa into subpopulations, integrity (carboxyfluorescein diacetate/propidium iodide staining), and functionality (hypo-osmotic swelling (HOS) test) of the spermatozoa plasma membrane were evaluated after thawing (T0) and after 30 min (T30) of incubation at 37 °C. There was no effect of SP addition on spermatozoa motility, but there was a significant positive effect on the kinematic parameters at T0 and T30. The addition of SP significantly increased the percentage of spermatozoa in the fast subpopulation at T0 as well as at T30. Plasma membrane integrity was not affected by the treatment, but functionality significantly decreased by 5% compared to the control group when samples were incubated for 30 min with A-SP. In conclusion, generally, the post-thaw addition of seminal plasma positively affected the post-thaw quality of semen from poor freezing stallions.

Genetic variants in male specific region (MSY) of ZNF280BY gene and their association with semen quality traits in Murrah buffalo bulls

Bull fertility is an important factor for genetic improvement; therefore prediction of fertility an early age of the bulls by using genetic markers is a goal for livestock breeding. Zinc finger protein280BY linked gene is predominantly expressed in testis and has a major role in spermatogenesis. An investigation was carried out to detect the genetic variants in the male specific region (MSY) of ZNF280BY gene and their association with semen quality traits in Murrah buffaloes. Polymerase Chain Reaction-Single Strand Conformation Polymorphism technique (PCR-SSCP) and DNA sequencing methods revealed three different SSCP band patterns (AA, BB, CC); one transition (C10375T) and an indel (insertion of T at 10460-10461). The associations of the genetic variants with semen volume, sperm concentration, Hypo Osmotic Swelling Test (HOST) in fresh and frozen semen were observed. AA and CC genotype bulls had high volume (P£0.05) of semen per ejaculation than BB genotype bulls. BB genotype bulls had high sperm concentration (106/mL) per ejaculate in comparison to AA and CC genotype bulls (P£0.05). AA and BB genotype bulls had a higher per cent of HOST (P£0.05) reacted sperms of fresh semen and per cent acrosomal integrity of frozen semen in comparison to CC genotype bulls. The observed association between SSCP variants in MSY region of ZNF280BY gene with semen quality parameters indicated the possibilities of using ZNF280BY as a candidate gene for identification of markers for semen quality traits in Murrah buffaloes.

A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa.

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p>0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.

Characteristics of the post-thawed Balinese bull semen extended in three different extenders and equilibration times

The objectives of the present study were to compare and determine the best post-thawed characteristics of balinese bull sperm cryopreserved in three different extenders; animal based (Tris-clarified egg yolk (Tris-cEY)), and non-animal based extenders (Bioxcell® (lecithin based) and Optixcell® (liposome based)) in combination with three different equilibration times (30 minutes, 2 hours, 4hours). Thirty six ejaculates were collected from six Balinese bulls and frozen in three extenders (Tris-cEY, Bioxcell® and Optixcell®) after equilibration in three different times (30 minutes, 2hours and 4hours). Computer-assisted sperm analysis (CASA), hypo-osmotic swelling test (HOST) and eosin nigrosin staining were used in the post-thawed semen analysis. There was a significant interaction between equilibration time and extender type for sperm motility, viability and membrane integrity. Thirty minutes equilibration time had the lowest values (P&lt;0.05) for all the evaluated parameters independent of extender type. Overall, semen extended in Tris-cEY, Bioxcell® and Optixcell® were similarly better when equilibrated at 4 hours (P&gt;0.05). Moreover, post-thawed semen which were extended in Optixcell® for 2 hours equilibration showed a better motility compared with the other extenders (P&lt;0.05). In conclusion, two hours equilibration of semen with Optixcell® is sufficient for semen freezing. Four hours equilibration has the best sperm survival, independent of the extender type.

PERBANDINGAN KUALITAS SPERMATOZOA SAPI JANTAN PASCA THAWING DENGAN PENGENCER YANG BERBEDA

The aim of this study was to evaluate the effects different extenders on motility, membrane and acrosome integrity of post-thawed bull sperm. Twenty ejaculates from four crossbreed bulls were collected. Within 5 min after sperm collection the fresh semen was divided into three groups based on types of extenders; (1) with Tris buffers, (2) with sodium citrate, and (3) with TALP extenders. A computer assisted semen analysis, hypo-osmotic swelling test, and fluorescence isothiocyanate labelled peanut agglutinin (FITC-PNA) techniques were used to determine motility, membrane integrity and acrosome integrity of spermatozoa, respectively, in both fresh ejaculate and frozen thawed semen. The percentage motility, membrane integrity and acrosome integrity in fresh ejaculate were higher than post-thawed. The sperm motility and membrane integrity were no significantly different (P&gt; 0,05) among all extenders. Whereas, the membrane acrosome integrity of spermatozoa in post-thawed was significantly different (P&lt; 0.05) when TALP extender was compared with sodium citrate, and Tris buffer extenders. In conclusion, all extenders resulted in a better sperm motility, membrane integrity after, while acrosome integrity was no significant after cryopreservation.

Determination of the relationship between percentage of hypo-osmotic swelling test scores and antisperm antibody assay in infertile patients after IUI technique

Determination of the relationship between percentage of hypo-osmotic swelling test scores and antisperm antibody assay in infertile patients after IUI technique

Clinical Correlation of Prostatic Calculi With Semen Parameters in Adult Men With Fertility Intention.

To investigate the correlation of prostatic calculi (PC) with semen parameters in men with fertility intention, this retrospective case–control study enrolled 1,303 participants ranging from 20 to 59 years old; 725 were diagnosed with PC using abdominal ultrasonography. Patients with PC were classified into the type A calculi group (discrete and small echoes) and type B calculi group (coarse and large masses of multiple echoes). Five hundred and seventy-eight men without PC were recruited for the control group. The clinical data of each group were collected and analyzed. The total motility was significantly lower for subjects with type B calculi (41.84% ± 17.50%) than for subjects in the type A calculi (51.78% ± 20.84%; p < .001) and control (54.47% ± 20.74%; p < .001) groups. The percentage of progressively motile was significantly lower for the type B calculi (31.66% ± 14.68%) group than the type A calculi (40.17% ± 17.09%; p < .001) and control (41.83% ± 17.05%; p < .001) groups. The results of the hypo-osmotic swelling test yielded significantly lower percentages in the type B calculi group (59.88% ± 17.13%) than the type A calculi (65.28 ± 14.43%; p = .005) and control (66.92 ± 16.12%; p < .001) groups. The type B calculi group had a significantly higher percentage of round cell concentration than control (4.5% vs. 1.0%; p = .007) did. Small and discrete PC may not influence semen quality among adult men with fertility intention, but larger and coarser PC are associated with decreased sperm motility.

Concurrence of HOST to some conventional sperm quality parameters and seminal enzymes of Jersey bulls semen

In this study, hypoosmotic swelling test (HOST) was correlated with sperm quality such as motility, acrosome integrity, morphological abnormality and seminal enzymes including AST, ALT and ALP. Results revealed positive concurrence of HOS test to motility and acrosomal integrity and negative correlation between ALP and ALT in both the freezable and nonfreezable ejaculates. Since the HOS test is quick, easy and inexpensive method so it can be deployed as routine semen assessment procedure in semen stations to differentiate between the freezable and non-freezable bull semen.

Effect of azolla supplementation in feed on semen freezability in bucks

The present study was conducted to evaluate the effect of azolla supplementation on semen freezability in Barbari bucks. Ten adult Barbari bucks (2–4 years old) were selected, and divided into control and treatment group (5 bucks in each group) as per completely randomized design. Bucks of control group were fed with 400 g concentrate pellet/day along with 6–7 h grazing while bucks of treatment group were supplemented with 100 g fresh azolla along with 400 g concentrate pellet and 6–7 h grazing. Semen ejaculates (8) were collected from each buck of control and treatment groups after 60 days of azolla supplementation. Immediately after collection, semen samples were evaluated for colour, volume, density, mass motility, progressive motility, live sperm count, acrosomal integrity and hypo-osmotic swelling positive spermatozoa. Then semen samples were diluted with tris–citric acid fructose diluents having 6% (v/v) glycerol as cryoprotective agent and 10% (v/v) egg yolk. After equilibration, semen samples were filled in straws and vapour frozen for 10 min 4 cm above the liquid nitrogen and finally stored into liquid nitrogen container. Post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo-osmotic swelling test were conducted to check the effect of azolla feeding on the freezability. Post thaw motility, live sperm count, acrosomal integrity and hypo-osmotic swelling positive spermatozoa were significantly higher in treatment group. It could be concluded that dietary azolla supplementation to breeding bucks improved the quality and freezability of semen favouring the use in artificial insemination program.

Progesterone decrease plasma membrane in human sperm with subnormal hypoosmotic swelling test scores

BackgroundProgesterone (P4) is known as a female hormone affecting oocyte maturation and developing uterine wall. A proteomic study identified several receptors including P4 receptors on human sperm. The role of P4 in human sperm cells remains unknown as to whether P4 has non-genomic effects on human sperm. The present study aims to determine the effect of progesterone (P4) on the hyperactivated motility and membrane integrity of human sperm cells.MethodsSemen from normal individuals was obtained from donors. The semen was washed by gradient density centrifugation. P4 was added to each semen sample to final concentrations of 0 (control), 250, 500, 750 and 1000 ng/mL. After the sample treatment was completed, the sperm membrane integrity was assessed with the hypoosmotic swelling test (sodium citrate dihydrate and D-fructose) and the hyperactivated sperm motility parameter was determined with the Computer Assisted Sperm Analyzer [CASA] (Hamilton Thorne, IVOS II, USA). The percentage was then compared between the treatment groups and the control group. The percentage differences were analyzed with the Sigmastat version 2.0 statistical program.ResultsAdministration of P4 increased sperm hyperactivated motility when compared with the control group at a concentration of 500 ng/mL, but the increase was statistically not signicant (p&gt;0.05). In contrast, P4 decreased sperm membrane integrity significantly (p=0.042). And the mean of plasma membrane integrity in all groups was subnormal hypoosmotic swelling test score. ConclusionProgesterone administration tends to increase sperm hyperactivated motility. The integrity of plasma sperm membrane was affected by progesterone.

EFFECT OF DIFFERENT CRYOPROTECTANTS ON THE POST-THAW SPERM CHARACTERISTICS AND IN VIVO FERTILITY OF BUFFALO (Bubalus bubalus) BULL SEMEN

This study aimed to investigate the effect of different cryoprotectants, glycerol (GLY) or ethylene glycol (EG) or dimethyl sulfoxide (DMSO) on sperm characteristics, and in vivo fertility of frozen-thawed buffalo-bull semen. A total of 85 ejaculates collected by artificial vagina from buffalo-bulls of proven fertility were used in this study. The collected ejaculates were examined for volume, motility, viability, morphology, and sperm cell concentration. The qualifying ejaculates (≥ 3 mass motion, &gt; 70% progressive motility and viability, &lt; 15% abnormal morphology and &gt; 1 x 109 sperm cells/mL) were pooled and diluted with Tris-based diluent containing either 7% GLY or 5% EG or 5% DMSO. After 4 h equilibration time, the diluted semen was loaded in 0.5 mL straws, labeled, sealed and frozen stored until analysis. Frozen straws were thawed and evaluated for progressive motility, viability, hypo-osmotic swelling test (HOST), acrosomal membrane integrity, and acrosome reaction (AR) in response to calcium ionophore A23187. Moreover, in vivo fertility was calculated after artificial insemination (AI) of 75 buffalo-cows (25 female/cryoprotectant) treated with double doses of prostaglandin F2α (PGF2α) 11 days interval. The proportions of progressive motility, viability and intact-acrosome were higher (p &lt; 0.05) in extender containing 7% GLY compared to 5% EG and 5% DMSO. The proportion of intact-plasma membrane was comparable (p ≥ 0.05) between GLY and EG but higher than that of DMSO. A time-dependent increase in the % AR and % relative AR was recorded in the three cryoprotectants with clear significant (p &lt; 0.01) difference among them at 30 and 60 min incubation, respectively. Moreover, GLY yielded higher pregnancy rate (52%) than EG (32%) and DMSO (16%). In conclusion, GLY is recommended for preservation of buffalo-bull semen in order to maintain sperm plasma membrane integrity and improve in vivo fertility of frozen-thawed buffalo-bull semen.Key words: buffalo-bull; frozen-thawed semen; cryoprotectant; estrus synchronization; In vivo fertility

Enhancement of Extender Excellence of Frozen Bull Semen Using α-Tocopherol as an Antioxidant

Background: Addition of the antioxidant to extender media is the most appropriate attempt to reduce structural losses encounter during the process of cryopreservation. Hence semen excellence could be maintained for longer duration without adverse impact. Additionally antioxidants are not only capturing the reactive oxygen species but also improve the sperm quality indicators and fertility. Accordingly, current elucidation has been executed to explore the dose depended appraisal of varied concentration of α-tocopherol in Tris-based extender on frozen-thawed bull semen quality parameters for enhancement of bull semen cryopreservation in the subtropical ecosystem of Peshawar.Materials, Methods &amp; Results: Experiments were carried out on semen that has been collected from both Achai-an indigenous breed and Holstein Friesian (HF) - the exotic breed in artificial vagina maintained at 42°C from the experimental bulls of either breed and processed independently breed wise. Semen specimens with above 70% motility were evaluated separately breed-wise under same environmental condition. Standard procedure was adopted to extend the collected semen in the experimental extenders and frozen subsequently. After thawing, further Analysis of the frozen straws of semen was carried out for sperm excellence indicators that include motility, viability, acrosomal integrity and functional integrity of spermatozoa under the subtropical condition. Sperm viability and acrosomal integrity were determined by dual staining procedure i.e. Trypan-blue and Giemsa stains. The hypo-osmotic swelling (HOS) test was used to assess plasma membrane integrity. The current elucidation demonstrated that α-tocopherol 1.5 mg/mL supplemented in extender had significantly (P &lt; 0.05) increased sperm excellence gauge that includes motility, viability, acrosomal integrity and functional membrane integrity in both the breeds. On the other hand, the result further elucidated those concentrations higher or lower than 1.5-1 mg/mL α-tocopherol supplemented in the present study resulted in a lower semen functional attributes subsequent to freezing.Discussion: Biological samples were preserved by various methods such cryopreservation at low temperature to maintain them intact and facilitate downstream experiments. Antioxidants were associated with the chemical breakage of the substrate consequential from oxidation and counterbalance the free radicals thus diminish the damaging impacts to spermatozoa in cryopreservation. There is lack of data with respect to lipid peroxidation and cell reinforcement limit in cryopreserved semen, and cryopreservation is related with the generation of receptive oxygen species (ROS) which lead to lipid peroxidation (LPO) of sperm membranes, bringing about lost motility, viability and fertility of sperm. Recently, in the reproductive management of the dairy animals, an important focus of research is to overcome the deleterious impact associated with semen cryopreservation. Elucidation of differential dose depended expression of α-tocopherol in semen extender has been evident in this study. The results were corroborated with diverse mammalian species under varied ecological management setups. Furthermore the detrimental impact of high doses of α-tocopherol in the semen extender has been demonstrated. It was deduced from the current finding that addition of α-tocopherol via dose depended approach in Tri extender enhanced the cryosurvivability of both Achai indigenous breed and HF the exotic breed, under existing reproductive management in the local ecosystem of Peshawar.

Effect of Mare Colostrum in Extenders for Freezing Stallion Semen

This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodine (PI) stain, acrosome integrity by fluorescein isothiocyanate and peanut agglutinine (FITC-PNA) and PI stain, plasma membrane functionality by hypo-osmotic swelling (HOS) test, and DNA denaturation by acridine orange (AO) test. There were no significant differences in the percentages of total motility, acrosome integrity, and DNA fragmentation among the extenders after thawing. Kinematics parameters showed significantly higher values in BotuCrio than in lactose extenders (P < .05). BotuCrio and lactose colostrum extender yielded significantly better rates for HOS-test, linearity, straightness, and wobble than egg-yolk extender (P < .05). However, in relation to sperm viability, lactose egg yolk extender showed significantly better results in comparison to the others seminal experimental media (P < .05). In conclusion, the incorporation of mare colostrum into cryopreservation media protected the sperm against cold-shock; therefore, it may be a good cryoprotectant agent alternative in extenders for freezing stallion semen.

Correlation between the Hypoosmotic SwellingT and DNA Fragmentation Assessed by the TUNEL Assay in Asthenozoospermia

Objective: To study the correlation between the hypoosmotic swelling test (HOST) and DNA fragmentation in asthenozoospermia assessed by the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) assay. Methods: This cross-sectional study was conducted in 27 semen samples obtained from infertile men with asthenozoospermia. Both HOST and TUNEL assay were performed for each sample. The sperm swelling pattern and positive apoptosis staining of individual spermatozoa were evaluated. HOST and TUNEL scores, and the proportion of positive staining in each grade were calculated in each sample. Results: The results showed a negative correlation between HOST and TUNEL scores (r = -0.428, P = 0.026). Sperm swelling grade A had a higher incidence of positive apoptosis staining when compared with other grades (P < 0.01). There was no statistically significant difference in positive apoptotic staining between other grades; nevertheless, sperm swelling grade D tended to have a lower incidence of positive apoptosis staining. Conclusion: Based on the results of this study, HOST may be used as an optional test to identify DNA-intact spermatozoa whereby sperm with a grade D swelling pattern should be selected preferentially for intracytoplasmic sperm injection (ICSI), whereas sperm with a grade A swelling pattern should be avoided for ICSI.