Animal Reproduction Science

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1 Animal Reproduction Science 123 (2011) Contents lists available at ScienceDirect Animal Reproduction Science journal homepage: Effect of transportation temperature on the quality of cauda epididymal spermatozoa of ram F.A. Lone, R. Islam, M.Z. Khan, K.A. Sofi Division of Animal Reproduction, Gynaecology and Obstetrics, Faculty of Veterinary Sciences and Animal Husbandry, S. K. University of Agricultural Sciences and Technology (K), Shuhama, Srinagar , Kashmir, India article info abstract Article history: Received 4 October 2009 Received in revised form 4 October 2010 Accepted 18 October 2010 Available online 4 November 2010 Keywords: Cauda epididymides Ram Sperm quality Storage Transportation temperature The objective of this study was to develop a protocol for ram epididymal sperm preservation that could be applied to wild ruminants for collection and preservation of spermatozoa from dead or hunted animals. Ram testicles collected from abattoirs were used to study the effect of two transportation temperatures viz. ambient temperature (AT) and refrigeration temperature (RT) on the cauda epididymal sperm quality at recovery and during preservation up to 72 h at 4 C. For AT the testicles were transported in normal saline in a container ( C) where as for RT the testicles were transported in an ice-chest (4.9 6 C). The results of the current study revealed that intact acrosome was significantly higher (P < 0.01) and other quality parameters like sperm motility, live sperm count, sperm concentration and major sperm abnormalities were also higher (P > 0.05) for RT than AT. The mean percent sperm motility for RT and AT was 81.67% and 78.33%, respectively. The corresponding figures were 92.08% and 90.46% for mean live sperm, 98.33% and 90.50% for intact acrosome, 0.50% and 0.33% for major sperm defects. The percent minor abnormality was 79.50% for RT and 77.67% for AT. The most prevalent minor defect was distal cytoplasmic droplet (70 80%). The mean sperm motility for RT and AT at 0 h was 82.50% and 75.00%, respectively and the corresponding values at 72 h of preservation were 60.00% and 45.83%. The mean live sperm at 0 h for RT and AT were 92.92% and 88.92%, respectively and the corresponding figures at 72 h were 81.50% and 73.17%. The mean intact acrosome at 0 h for RT and AT was 98.58% and 90.58%, respectively and at 72 h the corresponding values were 91.66% and 82.25%. The sperm motility, live sperm count and intact acrosome decreased significantly (P < 0.05) from 0 h to 72 h of preservation for both transportation temperatures. The sperm motility, live sperm count and intact acrosome also varied significantly between the transportation temperatures. The major sperm abnormality for both RT and AT at each hour of preservation up to 72 h was less than 0.5%. The study concluded that epididymides or testicles should be transported to the laboratory at RT (4.9 6 C) either in an ice-chest or portable refrigerator for their processing, evaluation and storage Elsevier B.V. All rights reserved. 1. Introduction Corresponding author. Present address: Animal Reproduction Division, Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly , UP, India. Tel.: ; fax: address: rafiqvet@gmail.com (R. Islam). The interest in preserving endangered species and valuable genetic material has resulted in increased attention towards possible recovery of viable sperm from the epididymides of dead animals (Foote, 2000). One goal of /$ see front matter 2010 Elsevier B.V. All rights reserved. doi: /j.anireprosci

2 F.A. Lone et al. / Animal Reproduction Science 123 (2011) conservation is to maintain healthy, genetically diverse, animal and plant populations. Genome Resource Banks can contribute to this, by providing a source of genes that can be infused into small or fragmented populations and thus counter the effects of unnatural selection pressures, genetic drift and inbreeding depression (Wildt et al., 1986, 1997; O Brien et al., 1983; O Brien, 1994). Rather than transporting stress-sensitive wild animals from one site to another, genetic heterogeneity could be maintained by shipping gametes or embryos. It may also be feasible to artificially inseminate free-living females with sperm from males of other wild, captive or hunted populations (Wildt et al., 1997) or from slaughtered or dead animals. With an increased interest in managing exotic animal species by Zoo s, Sanctuaries and/or private animal refuges, there is a growing concern over an increased inbreeding coefficient among the animal populations. Decreasing numbers of endangered and threatened species in the wild state is also a matter of concern for breeders and Zoo managers. To address the problem of inbreeding, it would be beneficial to develop an effective and plausible method to harvest and store epididymal sperm at postmortem for subsequent use in new assisted reproductive technologies (ART). The research finding of Foote (2000) in respect of motile sperm recovery from the epididymides of bulls, boars, rams and stallions after h of death has set a path for the storage of epididymal spermatozoa in farm animals. It is however, important to establish standard protocol to maintain sperm viability within the epididymides of dead animals during their transport to the laboratory as the researchers doing field work with endangered species usually do not have access to a well equipped laboratory. The ability to recover and store postmortem epididymal sperm will allow the use of valuable gametes from breeding male animals that either die or are slaughtered. The epididymal sperm preservation will also allow the use of valuable germplasm from males who have lost libido or having reproductive tract injury (Guerrero, 2006). Spermatozoa present in the testis or in the caput epididymis are immotile and immature, while those that reach the cauda are generally motile and mature and are thought to be capable of fertilization (Toshimori, 2003). The cauda epididymidis has been shown to be a source of viable mammalian sperm that are capable of fertilization and have resulted in normal births. Birth of a single Spanish ibex offspring (Capra pyrenaica hispanica) by artificial intrauterine insemination with cauda epididymal spermatozoa has been reported. This serves as an example of the practical use of epididymal spermatozoa for conservation of endangered species (Santiago-Moreno et al., 2006). Perusal of available literature revealed no systematic studies on the transportation of epididymidis and preservation of ram cauda epididymal spermatozoa. The present study was undertaken to study the effect of transportation temperature on the cauda epididymidis on the quality of spermatozoa at recovery and during their subsequent storage at 4 C. 2. Materials and methods 2.1. Collection of testicles The testicles used for this study were collected as early as possible from the slaughtered rams of 2 3 years of age from two abattoirs in Srinagar city. Some of the testicles were transported in normal saline at ambient temperature (AT; C) in a plastic container and some of them were transported at refrigeration temperature (RT; C) in an ice-chest to the laboratory. The duration of transportation from both the abattoirs was similar for both the treatment groups. The distance of both the slaughter houses from the laboratory is nearly equal. The transportation duration was 3 h. If occasionally the sample was brought to the laboratory before time due to variation in transport facility, then the uniform duration of 3 h was maintained by keeping the testicles at the same temperature and environment at the laboratory at which the sample is transported before their further processing Biometric analysis of testicles At the laboratory the testicles were measured, including testicular weight, cauda epididymal weight and diameter. The testicular weight was determined by electronic balance. After determining the testicular weight, the connective tissue around the testicle was removed with the help of a scissor. The testicles weighing more than 100 g were only processed. A total of 12 testicles from the ambient temperature (AT) and 12 testicles from refrigeration temperature (RT) transportation were selected for this study. The epididymis was separated from the testicle and weighed. The cauda epididymidis was then separated from whole epididymis. The weight and diameter of cauda epididymidis were recorded using electronic balance and vernier calliper, respectively Recovery of spermatozoa A total of 24 cauda epididymides comprising 12 for each transportation temperature were processed to compare the quality and quantity of spermatozoa recovered from the cauda epididymidis transported at two transportation temperatures. Incision method was applied to recover spermatozoa from the cauda epididymidis. The cauda epididymides were excised aseptically from the testis. Several longitudinal incisions (5 7) were made on the distal end of the cauda to expose the spermatozoa to the outer environment. Each cauda epididymidis was placed on a 35 mm Petri dish for 20 min to allow the sperm to swim out into the 3 ml of 2.9% sodium citrate buffer. Finally an additional 1 ml of buffer was used to rinse spermatozoa from the incised part of cauda epididymidis. The total cauda epididymal sperm out put was determined by conventional method (Haemocytometer method) Preservation of sperm samples Out of the 12 samples in each transportation temperature 6 samples were selected on the basis of percent

3 56 F.A. Lone et al. / Animal Reproduction Science 123 (2011) Table 1 Effect of transportation temperature on the characteristics of cauda epididymal spermatozoa (mean ± S.E.M.). Transportation temperature Parameters studied Sperm motility (%) Live sperm (%) Intact acrosome (%) Sperm concentration ( 10 6 ) Sperm abnormality Major (%) Minor (%) Total (%) AT ± ± a ± ± ± ± ± 2.22 RT ± ± b ± ± ± ± ± 0.48 Overall ± ± ± ± ± ± ± 2.46 AT ambient temperature; RT refrigeration temperature. Means in a column with different superscripts (a and b) differ significantly (P < 0.01). progressive motility after sperm recovery. Six sperm samples showing 70% motility were extended with egg yolk citrate (EYC) extender (2.9% sodium citrate buffer 80 ml, egg yolk 20 ml, penicillin G sodium 80,000 i.u. and streptomycin sulphate 100 mg) at the rate of 1:2 (4 ml sperm sample with 8 ml extender) for each transportation temperature and preserved up to 72 h at 4 C Quality of spermatozoa The quality of spermatozoa was determined by measuring % motility, % live sperm, % abnormalities and % intact acrosomes. The percentage of progressively motile spermatozoa was estimated following the method described by Zemjanis (1970). A drop of sperm sample was placed on a grease free slide maintained at 37 C on biotherm and a coverslip was put over the drop and examined under high power objective (40 ) of a microscope. Percentage of live sperm count in the sperm samples was determined using the nigrosin eosin combined stain as per the method described by Blom (1977). Morphological abnormalities were also studied on the slide prepared for live sperm count. The sperm abnormalities were divided into major and minor sperm defects. A total of 200 sperm cells were examined and out of these percent major sperm defects was calculated (Blom, 1977). The percent of intact acrosomes was determined in the sperm samples as per the method described by Watson (1975). Geimsa stain powder (3.8 g) was ground with 375 ml of methanol (AR Grade) with the help of pestle and mortar. The mixture was transferred to a 500 ml reagent bottle; evaporated volume of methanol was replaced and 125 ml of glycerol was added to it. This stain mixture was kept for ripening in an incubator at 37 C for one week. During the period of ripening the bottle was shaken daily for few minutes. The Hancock s Fixative is prepared by dissolving sodium chloride 10 g, sodium bicarbonate (NaHCO 3 ) 0.5 g, formalin 125 ml in 1000 ml distilled water. Sorensen s phosphate buffer was prepared by mixing 17 ml of 0.1 M KH 2 PO 4 solution with 33 ml of 0.1 M Na 2 HPO 4 solution. The ph was adjusted using a digital ph meter. Geimsa working solution was prepared by mixing 3 ml Geimsa stock solution, 2 ml Sorensen s phosphate buffer and 35 ml distilled water in a coupling jar Staining procedure A small drop of each sperm sample was placed on a grease free slide and a smear was prepared. The air dried smears were placed in Hancock s fixative for min in coupling jar. After fixation of the slides, washing was done under slow running tape water for another min and washed with distilled water. Finally the slides were placed in coupling jar containing Geimsa working stain and kept for overnight. On next morning the slides were removed, washed with slow running tape water and finally with distilled water, air dried and was observed under oil immersion objective (100 ). A total of two hundred spermatozoa were counted and percentage of intact acrosome was calculated Statistical analysis The data in respect of characteristics of cauda epididymal spermatozoa immediately after transportation and subsequent recovery were analyzed by using nonparametric Mann Whitney test for comparison between the two transportation temperatures (Table 1). The data pertaining to sperm quality during preservation was analysed by two factor analysis of variance (ANOVA) for comparison between transportation temperatures and between different hours of preservation with the help of Statistical Software SPSS Version-13 (SPSS Corporation, USA). The data pertaining to percentage major sperm abnormality was first transformed by using square root transformation (X + 0.5) 1/2. Mean square values of percentage major sperm abnormality are the values of the transformed data (Table 4). All the data are expressed as mean ± S.E.M. (Tables 1 3). The level of significance was set at P < Results The effect of the two transportation temperatures on sperm motility %, live sperm %, intact acrosome %, sperm concentration (millions) and sperm abnormalities % (major and minor) in sperm harvested from cauda epididymides are presented in Tables Biometry of testicles and epididymis The testicular weight (g), cauda epididymal weight (g) and cauda diameter (cm) for AT and RT were and 140.3, 6.60 and 7.44, and 2.18 and 2.36, respectively. However, the parameters did not differ significantly between the two transportation temperatures.

4 F.A. Lone et al. / Animal Reproduction Science 123 (2011) Sperm quality at recovery Table 2 Effect of transportation temperature on the sperm motility, live sperm count and intact acrosome at different hours of preservation (mean ± S.E.M.). Preservation period (h) Sperm motility (%) Live sperm (%) Intact acrosome (%) AT RT Overall AT RT Overall AT RT Overall ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.55 Overall ± ± ± ± ± ± ± ± ± 0.82 The sperm motility for RT (81.67%) was higher than AT (78.33%). The mean percent live sperm for RT (92.08%) was higher (P > 0.05) than the live sperm count obtained for AT (90.46%). However, the mean percent intact acrosome for RT (98.33%) was significantly higher (P < 0.05) than AT (90.50%). The sperm abnormality was mainly contributed by percent minor abnormality and accounted 77.67% for AT and 79.50% for RT (Table 1). The most prevalent minor defect was distal cytoplasmic droplet (70 80%) Sperm quality during preservation The mean percent sperm motility at 0 h for RT and AT was 82.50% and 75.00%, respectively and the corresponding values at 72 h of preservation were 60.00% and 45.83%. The mean percent live sperm at 0 h for RT and AT was 92.92% and 88.92%, respectively and the corresponding figures at 72 h were 81.50% and 73.17%. The mean percent intact acrosome at 0 h for RT and AT was 98.58% and 90.58%, respectively and at 72 h the corresponding values were 91.66% and 82.25%. The sperm motility, live sperm count and intact acrosome decreased significantly (P < 0.05) from 0 h to 72 h of preservation for both transportation temperatures (Tables 2 and 4). The sperm motility, live sperm count and intact acrosome also varied significantly between the transportation temperatures. The interaction between the transportation temperature (TT) and preservation periods (PP) was found to be non-significant for all the sperm quality parameters studied (Table 4). The transportation temperature and preservation periods have significant effect on the percentage minor sperm abnormality (<0.05); however, the effect was found to be non-significant for the major sperm abnormalities. The major sperm abnormality for both RT and AT at each h of preservation from 0 to 72 h was less than 0.5%. Overall minor sperm abnormality, irrespective of transportation temperatures, during the preservation hours was 84.81% (Table 3). However, the minor abnormalities were mainly consisting of distal cytoplasmic droplet (70 80%). 4. Discussion Collection and transportation of testicles of dead, hunted or slaughtered animals as early as possible to the laboratory is important to maintain sperm quality till processed. Otherwise, spermatozoa within the body of male animals degenerate quickly after death (Garde et al., 1998). Recovery and cryopreservation of epididymal spermatozoa would be one useful way to rescue the germplasm of these dead animals, and to use them to preserve endangered species. However, under field conditions it is not always possible to process and preserve epididymal spermatozoa quickly due to a lack of technicians and equipment nearby. Transportation of testicles to the laboratory to reduce tissue and cell death and thus maintain subsequent viability of the sperm inside the epididymides is essential for the recovery of good quality of spermatozoa. The values for quality parameters like sperm motility, live sperm count were non-significantly (P > 0.05) higher

5 58 F.A. Lone et al. / Animal Reproduction Science 123 (2011) Table 3 Effect of transportation temperature on the morphological abnormality at different hours of preservation (mean ± S.E.M.). Preservation period (h) Sperm abnormality (%) Major Minor Overall AT RT AT RT Major Minor ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.94 Overall 0.12 ± ± ± ± ± ± 0.95 Table 4 Analysis of variance of the percentage motile sperm, live sperm, intact acrosomes and sperm abnormality at different hours of preservation using two transportation temperatures. Source of variation d.f. Mean squares Sperm motility Live sperm count Intact acrosome Sperm abnormality (%) Major Minor Transportation temperature (TT) * * * a * Preservation period (PP) * * * a * TT PP a a 4.33 a a a Error a Non-significant. * P < and intact acrosome was significantly (P < 0.01) higher for the sperm samples obtained from cauda epididymidis transported at RT than AT (Table 1). Similar trend was also observed during the preservation of the sperm samples in EYC extender at 4 C up to 72 h. However, epididymides could be transported at both ambient (AT) and refrigeration (RT) temperatures for collection and preservation of spermatozoa as there was no significant difference in sperm motility and liveability studied and the values for sperm quality were higher than the permissible limit, but the quality deteriorated comparatively quickly for AT than RT as preservation time progressed. The sperm motility, live sperm count and intact acrosome were higher at each hour of preservation in EYC extender at 4 C in case of epididymal transportation at RT than at AT and the difference was significant (P < 0.05) (Tables 2 4). If spermatozoa are to be collected after some time due to lack of equipment or technician or collected immediately but intended to be stored at 4 C for some days, then transportation at refrigeration temperature is an excellent choice. Kaabi et al. (2003) also suggested that ram epididymides must be stored at 5 C for 24 h when epididymal spermatozoa could not be immediately collected and cryopreserved which is in agreement with our study. The live sperm count also higher for the refrigeration temperature (RT) transportation of epididymides at each hour of preservation from 0 to 72 h (Table 2). Ground transportation at room temperature proved to be unsuitable for optimal sperm recovery and subsequent preservation in stallion (Neild et al., 2006) also in line with the present study. The reason for higher motility in case of refrigeration temperature can be explained on the basis of reduced metabolic rate of spermatozoa inside the epididymides when transported at RT. The beneficial effect of refrigeration on various parameters of sperm quality, especially motility, may be due to the reduced metabolic rate of sperm cells when they are at 5 C(Salamon and Maxwell, 2000). Sankai et al. (2001) reported that motility of mouse epididymal spermatozoa decreased when the storage temperature was increased, suggesting that this effect was related to changes in spermatozoa metabolic activity. Martinez-Pastor et al. (2005) also found that membrane and acrosomal integrity of epididymal spermatozoa from wild ruminants was enhanced in postmortem conditions if cooling of the epididymides occurred. Epididymal environment provides the most ideal condition for sperm survival, however, refrigeration at 5 C is necessary to diminish energy wastage and extend sperm lifespan (Martins et al., 2009). No significant difference in terms of major sperm abnormalities was revealed between the two transportation temperatures and between the preservation periods from 0 to 72 h and. The major abnormalities were less than 0.5% for both transportation temperatures. The higher percentage of minor sperm abnormalities for both transportation temperatures is mainly contributed by distal cytoplasmic droplet (70 80%) which is considered as normal part of sperm maturation process during epididymal migration and does not interfere in fertilization process. Our study interpreted that though both the transportation temperature can be used for transportation of testicles and epididymidis to laboratory, but refrigeration temperature is the best for maintenance of higher sperm quality during transit till processing of the samples. 5. Conclusion Testicles or free epididymides of dead or slaughtered animals can be transported at atmospheric temperature in normal saline if spermatozoa are to be used immediately. However, refrigeration temperature is best for transporta-

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