Post-thaw Stage Research Articles

Sequestration of PDC-109 protein improves freezability of crossbred bull spermatozoa

A study was carried out to assess the effect of sequestration of PDC-109 protein, a majority constituent of heparin binding proteins (HBP) of seminal plasma, on freezability and in vitro fertilizing ability of crossbred bull spermatozoa after cryopreservation. The study consisted of isolation and characterization of PDC-109 protein to raise anti-sera against it in rabbits. Following which, raised antibodies against PDC-109 protein was quantitated and coated in tubes used for collection of ejaculates. Semen ejaculates thus collected were cryopreserved using EYTG extender. Physico-morphological characteristics, viz. motility, viability, acrosomal integrity and HOS response as an indicator of freezability of cryopreserved spermatozoa were determined at pre freeze as well as post thaw stage. At pre freeze stage, a significant (p<0.05) improvement in viability (83.83±2.18 vs 75.17±2.42) and acrosome integrity (81.33±2.38 vs 72.83±2.39) in antibodies treated group than control was observed. Similarly, increase in HOS responsive spermatozoa was highly significant (p<0.01) than control (78.83±1.69 vs 67.5±1.75). At post thaw stage, significant (p<0.05) improvement in viability (69.50±2.16 vs 60.33±2.19) and HOS responsive spermatozoa (68.67±1.62 vs 58.50±1.32) and highly significant (p<0.01) increase in individual motility (56.17±1.83 vs 47.00±1.86) and acrosome integrity (75.17±2.38 vs 61.83±2.1) was observed in antibodies treated group when compared to control was observed. The results from the study revealed that sequestration of PDC-109 protein from semen samples leads to significant improvement in pre-freeze and post-thaw values of above parameters in cryopreserved spermatozoa. It is thus concluded that sequestration of PDC-109 protein from ejaculates improves freezability of crossbred bull spermatozoa.

Computer assisted semen analysis for quantification of motion characteristics of bull sperm during cryopreservation cycle

Aim: The study was undertaken to quantify and to analyze the changes in the motion characteristics of bull spermatozoa during various stages of cryopreservation cycle. Materials and Methods: Using computer assisted semen analysis (CASA) technique, 26 ejaculates, collected from two Jersey bulls were analyzed for motility, head behaviour and swimming pattern of spermatozoa on dilution, pre-freeze and post-thaw stages of cryopreservation. French straw technique was employed for deep-freezing of semen using liquid nitrogen. O Results: Equilibration of diluted semen at 5 C has significantly (P< 0.01) reduced sperm motility, progressive motility, path velocity, and progressive velocity. Beat cross frequency was also affected significantly (P<0.05) by equilibration. Freezing and thawing processes drastically affected all the motility, velocity and head behaviour characteristics (P< 0.01). Conclusion: CASA facilitate objective evaluation sperm motion characteristics. Adoption of CASA technique has the potential for improvements in evaluation of semen thereby the quality of frozen semen for fertility can be enhanced. Artificial insemination (AI) in bovines is a classical assessment of quality of motility in terms of velocity, example for successful application of a biotech- swimming pattern, sperm head behaviour etc., may nological method at field level. Although fertility of help in better understanding of the possible sperm frozen-thawed semen is generally acceptable but the function. The advent of computer assisted semen analysis efficiency of cryopreservation is still relatively low. (CASA) has brought a new dimension to semen Rapid rate of cryopreservation results in cell damage evaluation. The CASA technique yields repeatable to spermatozoa due to osmotic imbalance (1). and highly reliable results on kinematics of ejaculates The mammalian spermatozoa acquire the capacity based on measurements of individual sperm cells. for flagellar movements during epididymal transport Therefore, assessment of sperm motility coupled with and it is modulated in the female reproductive tract kinetic measurements would help in better evaluation before termination during fertilization. These changes of semen quality. in flagellar activity reflect the physiological events within the sperm cell including the metabolic activity Materials and Methods and alteration of the sperm membrane (2). Further the Two Jersey bulls, aged 2 years, maintained for events of semen processing during cryopreservation instructional purpose for the students of Madras cycle bring injuries to sperm membrane leading to Veterinary College, Chennai under stall-fed conditions changes in the sperm physiology which may be were used for the study. The bulls were fed with 3 kg of reflected in sperm motility (3). Cryopreservation also concentrate/day/bull in addition to 3 kg of dry fodder affects the functional integrity of acrosome and per bull. Water and green fodder were provided ad mitochondria that is responsible for the generation of libitum. Since semen collection by artificial vagina energy from intracellular stores of ATP (4). method did not involve any intervention in the normal Conventionally, sperm motility estimation is done physiology of the animals, this experiment was by visual approximation of progressively moving exempted approval from Institutional Animal Ethics spermatozoa using phase contrast microscope. The

Effect of Recombinant Bovine Somatotropin on Plasma Concentrations of Insulin‐like Growth Factor I, Insulin and Membrane Integrity of Bull Spermatozoa

This study aimed to evaluate the effect of the exogenous recombinant bovine somatotropin (rbST) on plasma concentrations of insulin-like growth factor I (IGF-I), insulin and semen quality of bulls. Twenty bulls (Aberdeen Angus and Brangus) were divided by breed into two groups. Placebo group was injected with NaCl 0.9% (s.c.) and treatment group with rbST (s.c., 500 mg) at days 0 and 14 of the experiment. Immediately after semen collection, blood samples were taken on days 0, 14, 28, 42 and 56 of the experiment. Semen was also collected on day 70 of the experiment. Evaluation of sperm motility was performed at pre-freezing and post-thawing stage, whereas assessment of sperm membrane integrity was performed after freezing and thawing. Analysis of data revealed that the effect of treatment and treatment-by-collection day on plasma concentrations of IGF-I and insulin was not significant. However, mean plasma concentrations of IGF-I and insulin were affected (p < 0.0001) by days of blood sampling. Effect of treatment and treatment-by-collection day on motility of spermatozoa was similar (p > 0.05) at pre-freezing and post-thawing stage. Intactness of plasmalemma and tail membrane of spermatozoa at post-thawing stage was higher (p < 0.05) in rbST-treated group than in control. In conclusion, rbST did not affect plasma concentrations of IGF-I and insulin, however, it did improve post-thaw sperm membrane integrity.

Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP)

Previous cryopreservation studies with buffalo cauda epididymal spermatozoa have reported a deleterious effect of seminal plasma heparin binding protein (HBP). The amount of HBP used in these studies was meager compared to the normal level of HBP in the buffalo ejaculate, still the damage induced upon the spermatozoa was substantial when compared to that incurred to the spermatozoa during routine freezing of ejaculated semen. Thus there might be some factor(s) in the seminal plasma, which reduce the deleterious effect of HBP on spermatozoa during cryopreservation of ejaculated semen. This study was conducted to investigate for the presence of any such factor in buffalo seminal plasma. Seminal plasma proteins were separated on their heparin binding properties as heparin binding (HBP) and non-heparin binding (NHBP). The separated proteins were added to the extender of buffalo cauda epididymal semen for cryopreservation either alone or in combination. The spermatozoa were assessed for progressive motility, viability, acrosomal integrity and response to hypo-osmotic solution test (HOST) at prefreeze and post-thaw stages of cryopreservation. NHBP was found to provide some degree of protection to buffalo spermatozoa against cryopreservation stress as well as the deleterious effect of HBP during cryopreservation.

Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa

The study was conducted to assess the effect of heparin binding seminal plasma proteins (HBP) on freezability and in vitro fertilizing ability of buffalo cauda epididymal spermatozoa. Spermatozoal motility, viability and acrosomal integrity at prefreeze and post-thaw stages were studied. The in vitro fertilizing ability of spermatozoa was assessed by the application of two tests, i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic sperm swelling test (HOST). HBP isolated from buffalo seminal plasma and maintained in the laboratory were used for the study. Twelve pairs of epididymis from adult buffaloes slaughtered at the local abattoir were used for the study. The results indicated that HBP addition improved the progressive motility, BCMPT and HOST response at prefreeze level. HBP at a concentration of 40 μg/ml showed better results than HBP at a concentration of 80 μg/ml. However, subjecting the HBP treated spermatozoa to cryopreservation resulted in significant reduction of motility, viability, acrosomal integrity and response to BCMPT and HOST in the HBP treated groups when compared to those in control group. The deleterious effect of HBP was found to be concentration dependent with the higher concentration causing higher post-thaw damage.

Is Cryopreservation a Homogeneous Process? Ultrastructure and Motility of Untreated, Prefreezing, and Postthawed Spermatozoa of Diplodus puntazzo (Cetti)

This study subdivides the cryopreservation procedure for Diplodus puntazzo spermatozoa into three key phases, fresh, prefreezing (samples equilibrated in cryosolutions), and postthawed stages, and examines the ultrastructural anomalies and motility profiles of spermatozoa in each stage, with different cryodiluents. Two simple cryosolutions were evaluated: 0.17 M sodium chloride containing a final concentration of 15% dimethyl sulfoxide (Me2SO) (cryosolution A) and 0.1 M sodium citrate containing a final concentration of 10% Me2SO (cryosolution B). Ultrastructural anomalies of the plasmatic and nuclear membranes of the sperm head were common and the severity of the cryoinjury differed significantly between the pre- and the postfreezing phases and between the two cryosolutions. In spermatozoa diluted with cryosolution A, during the prefreezing phase, the plasmalemma of 61% of the cells was absent or damaged compared with 24% in the fresh sample (P < 0.001). In spermatozoa diluted with cryosolution B, there was a pronounced increase in the number of cells lacking the head plasmatic membrane from the prefreezing to the postthawed stages (from 32 to 52%, P < 0.01). In both cryosolutions, damages to nuclear membrane were significantly higher after freezing (cryosolution A: 8 to 23%, P < 0.01; cryosolution B: 5 to 38%, P < 0.001). With cryosolution A, the after-activation motility profile confirmed a consistent drop from fresh at the prefreezing stage, whereas freezing and thawing did not affect the motility much further and 50% of the cells were immotile by 60–90 s after activation. With cryosolution B, only the postthawing stage showed a sharp drop of motility profile. This study suggests that the different phases of the cryoprocess should be investigated to better understand the process of sperm damage.

Effect of cryoprotectants on certain seminal attributes and on the fertility of buck spermatozoa

Semen samples were obtained from 12 bucks (3 Beetal, 3 Black Bengal and 6 Beetal × Black Bengal) and 10 different extenders were constituted with varying concentrations of glycerol, DMSO, glycerol + DMSO and glycerol + lactose as the sperm cryoprotective agents. After the collection of semen samples, they were assessed for quality, diluted in different extenders after removal of seminal plasma, packaged in ministraws and frozen after equilibration (5′C) for 5 h. The samples were evaluated immediately after equilibration and again 24 h after freezing for progressive motility, percentage of live spermatozoa and acrosome, head and tail abnormalities. Both motility and the percentage of live spermatozoa were most affected by extenders containing only DMSO and these values improved in glycerol + DMSO extenders as the concentration of glycerol was increased while DMSO was decreased. However, these values were significantly higher in extenders containing glycerol + lactose as the cryoprotective agents, and were found to increase with increased concentration of lactose, being highest in TYGL 180. Acrosomal and tail abnormalities tended to increase between post equilibration and post thawing stage, and were higher in extenders containing the higher levels of DMSO. Significantly (P < 0.01) lower percentages of abnormalities were recorded in the glycerol + lactose extenders. The fertility results showed nonsignificant effect of extenders on the conception rate of does.