Recovery of epididymal spermatozoa from bull and red deer, stored at different times and temperatures before freezing - thawing

2012 ◽  
Vol 52 (8) ◽  
pp. 741 ◽  
Author(s):  
V. Malcotti ◽  
V. Pelufo ◽  
N. Bergamo ◽  
E. Aisen

In order to preserve male germoplasm, the recovery and cryopreservation of spermatozoa from the epididymides of hunted animals represents an accessible source of gametes. As a first experimental model, epididymal spermatozoa from slaughtered bulls were recovered at 30, 54, 78 and 102 h after death. The scrotal contents were stored at either 5 or 20°C. The sperm cells of each treatment (time + temperature combinations) were frozen with Triladyl (T) or Triladyl + Trehalose (TT) diluents. In order to assess sperm viability and integrity, post-thawing evaluation included individual motility, supravital stain, hyperosmotic swelling test (E+), acrosome status and sperm chromatin structure assay. Both at raw and post-refrigerated states, the sperm motility rate was higher in sperm obtained from epidydmes stored at 5°C, compared with those stored at 20°C for all collection times. Sperm collected at 102 h after death from epididymides stored at 5°C maintained a motility of 20% (120 h, raw state). When comparisons were carried out after thawing, motility was higher in the 5°C group, achieving the best results with TT diluent (7.5%) at 102 h. However, when supravital stain and E+ tests were observed, viability and membrane integrity were well preserved even at 102 h post mortem (30 and 36%, respectively, with TT diluent at 5°C). These results suggest that frozen-thawed epididymal spermatozoa could have a low motility rate while most of them remain alive. Acrosome status was not greatly affected by storage time. In a second experiment, epididymal spermatozoa from hunted red deer stags (Cervus elaphus) were recovered at 4 and 30 h after death. The scrotal contents were stored at 20°C, because that temperature is closer to field and shipment conditions. The sperm cells were frozen with TT diluent. Post-thawing evaluation included the same parameters indicated for bull spermatozoa. The assessment of spermatozoa collected at 30 hours post mortem and then subsequently frozen and thawed indicated that at this time an acceptable motility rate (35%) and viability (39.7%) were achieved. Frozen and subsequently thawed epididymal spermatozoa showed 47.9% of membrane integrity, 59.3% of acrosome integrity and 26.5% of chromatin damage, using TT diluent. A preliminary in vivo trial demonstrated that the pregnancy rate in artificially inseminated deer decreased when sperm were obtained at 30 h post mortem. According to these results, it may be concluded that storage at 5°C is better than 20°C to obtain well preserved epididymal spermatozoa from bulls, and that TT could be a useful cryoprotectant to preserve viable and fertile sperm cells after the freezing–thawing process. Before these results can be applied to assisted reproduction programs in endangered deer species, some adaptations must be developed.

2007 ◽  
Vol 19 (1) ◽  
pp. 183
Author(s):  
J. P. Soler ◽  
G. G. Kaiser ◽  
N. Mucci ◽  
L. B. Ferre ◽  
R. H. Alberio

Multiple ovulation and embryo transfer (MOET) programs for red deer (Cervus elaphus) have been established commercially over the last decade, with embryo cryopreservation being a related practice necessary to enhance the use of valuable genetic information. The aim of this work was to establish alternative methods for red deer embryo cryopreservation by using slow freezing with ethylene glycol (SF–EG) and vitrification by open pulled straw (OPS) methods. After surgical flushing of 18 superstimulated donors, 54 transferable embryos were recovered; 28 were transferred fresh to synchronized recipients and the others were cryopreserved by SF–EG (n = 11) or OPS (n = 15), respectively thawed or warmed, and transferred to recipients. Fresh embryos were maintained in Dulbecco's PBS + 20% cow serum (holding medium, HM) until transfer (maximum 3 h after collection). SF–EG cryopreserved embryos were suspended in HM + 1.78 M EG + 0.1 M sucrose + 4 mg mL−1 BSA. After a 10-min equilibration, embryos were loaded individually into 0.25-mL plastic straws and placed into a −7°C methanol bath chamber. After seeding (5 min later), the straws were cooled from −7 to −35°C at a rate of 0.5°C min. Straws were plunged into and stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 s; their contents were drained into HM until transfer. Embryos were vitrified using the OPS method with minor modifications. They were first incubated in HM + 1.78 M EG + 1.3 M DMSO for 3 min and then transferred for 25 s into a vitrification solution of HM + 3.56 M EG + 2.6 M DMSO + 0.5 M sucrose. Each embryo was loaded by touching a 1-µL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straws into HM + 0.25 M sucrose for 5 min. Embryos were then transferred into HM + 0.15 M sucrose for 5 min and finally to HM until transfer. Both types of cryopreserved embryos were transferred a few hours after collection, immediately after thawing or warming. Before embryo transfer, the presence of corpus luteum (CL) of recipients was confirmed by laparoscopic examination. Each embryo was surgically transferred into the apical extreme of the uterine horn ipsilateral to the CL of one recipient. Pregnancy was determined by ultrasonography 41 days after embryo transfer. The pregnancy rate between groups was compared with the chi-square test (P < 0.05). No statistical differences were found between groups (Table 1). Our results show that both vitrification and slow freezing methods with EG are suitable to cryopreserve red deer embryos. Table 1. Pregnancy rates in recipient hinds after transfer of fresh, vitrified, or frozen red deer embryos


2001 ◽  
Vol 49 (2) ◽  
pp. 223-227 ◽  
Author(s):  
Sz. Nagy ◽  
A. Kovács ◽  
T. Zubor ◽  
Z. Zomborszky ◽  
J. Tóth ◽  
...  

A simultaneous live/dead and acrosome staining, originally described for domestic mammals, was successfully applied on red deer (Cervus elaphus) and fallow deer (Dama dama) spermatozoa collected from the cauda epididymidis and vas deferens of shot stags. The staining is simple enough for routine application. Seven classes of spermatozoa were distinguished in the smears of frozen/thawed semen samples. Morphology, including cytoplasmic droplets, was evaluated as well. Percentage of live cells with intact acrosomes and with no other morphological aberrations might be a practical index of semen quality.


1999 ◽  
Vol 47 (2) ◽  
pp. 263-270 ◽  
Author(s):  
Z. Zomborszky ◽  
T. Zubor ◽  
J. Tóth ◽  
P. Horn

Sperm samples were collected from the epididymides of 11 hunter-killed stags (Cervus elaphus hippelaphus) within 2 to 17 h post mortem in September 1991. Progressively motile spermatozoa were diluted and deep-frozen in tris-yolk extender by a procedure routinely used for bovine semen. The pre-freezing motility of spermatozoa from 6 stags was higher than 80%, while the sperm of 5 animals was found to be unsuitable for dilution. In the post-thawed sperm of six stags 40-50% of the spermatozoa showed progressive motility and the number of viable spermatozoa ranged from 8.6 to 26.7 × 106 per 0.25 ml straw. Two years later, three hinds were superovulated by the use of a progesterone-releasing intravaginal device (CIDR type G, Carter, Holt Harvey Plastic Products Group Ltd., Hamilton, New Zealand) for a period of 14 days and with follicle stimulating hormone (Folicotropin inj., Spofa, Prague). Each hind was inseminated artificially 60 h after the withdrawal of CIDR with thawed sperm injected into the uterus via the vagina. Seven days later the uteri were flushed out, as a result of which 3 early blastocysts + 1 ovum, 3 morulae + 4 ova, and 1 morula + 7 ova, respectively, were recovered from the three hinds. Deer embryos were frozen according to a glycerolbased freezing protocol. A further two years later two hinds were oestrussynchronised with CIDR type G and 300 IU PMSG (Folligon inj., Intervet, NL), and two of the thawed embryos were transplanted into two recipient hinds 7 days after heat. One of these gave birth to a normal stag fawn in June 1996. This was the first deer born in Hungary from embryo transfer. The results obtained indicate that sperm from top stags shot in the course of hunting can prove useful for the preservation of genetic material or in the development of the farmed deer system.


2003 ◽  
Vol 24 (5) ◽  
pp. 746-756 ◽  
Author(s):  
Ana J. Soler ◽  
Andrés J. García ◽  
María R. Fernández-Santos ◽  
Milagros C. Esteso ◽  
José J. Garde

2007 ◽  
Vol 67 (4) ◽  
pp. 738-753 ◽  
Author(s):  
M.R. Fernández-Santos ◽  
F. Martínez-Pastor ◽  
V. García-Macías ◽  
M.C. Esteso ◽  
A.J. Soler ◽  
...  

2020 ◽  
Vol 41 (1) ◽  
pp. 181
Author(s):  
Patrícia Andrade dos Santos ◽  
Vânia Maria França Ribeiro ◽  
Augusto Luiz Faino Alves ◽  
Vanessa Lima da Silva ◽  
Breno Kalyl Freitas Nascimento ◽  
...  

The objective of this study was to evaluate the morphology, morphometry, and membrane integrity of epididymal spermatozoa of spotted pacas using spermatic cells collected from the epididymal tails of five animals. The flotation method using the ACP-123® and Botusemen Special® extenders was performed, and samples were stained in Diff-Quick and eosin-nigrosine. Descriptive statistics of data were obtained and Student’s t-test was performed. The morphology of 200 Diff-Quick-stained spermatozoa showed that they had an oval head with three vesicles in the acrosomal region, a midpiece, an elongated tail; moreover, 27% of the spermatozoa exhibited cellular defects. The morphometry of 100 sperm cells (analyzed with an optical microscope and the EZ Leica LAS software for Windows) presented the following measurements (mean ± SD): total length 43.87 ± 4.91 ?m, head 7.54 ± 0.82 ?m, midpiece 5.35 ± 0.83 ?m, tail 30.72 ± 2.55 ?m, and head width 5.30 ± 0.68 ?m. Of the 2,000 cells stained with eosin-nigrosine for membrane integrity evaluation, 83.8% diluted in ACP-123® and 72.9% diluted in Botusemen® had intact membranes. The results of this study suggest that epididymal spermatozoa of pacas can be used in assisted reproduction programs; moreover, our study adds knowledge to the reproductive biology of wild animals, and encourages further research on the role of the three acrosomal vesicles present in this species.


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