Paweena Thuwanut

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.

Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm.

The objectives were to determine the: 1) extent of epithelial and red blood cell contamination in epididymal cat sperm samples recovered by the cutting method; 2) efficacy of simple washing, single-layer centrifugation (SLC), and swim-up for selecting epididymal cat sperm; and 3) effects of freezing and thawing on cat sperm selected by various techniques. Ten unit samples were studied; each contained sperm from the cauda epididymides of four cats (total, approximately 200 x 10(6) sperm) and was equally allocated into four treatments: 1) simple washing, 2) single-layer centrifugation through colloid prior to cryopreservation (SLC-PC), 3) single-layer centrifugation through colloid after cryopreservation (SLC-AC), and 4) swim-up. Centrifugation (300 x g for 20 min) was done for all methods. The SLC-PC had a better recovery rate than the SLC-AC and swim-up methods (mean+/-SD of 16.4+/-8.7, 10.7+/-8.9, and 2.3+/-1.7%, respectively; P<0.05). The SLC-PC, SLC-AC and swim-up samples contained less red blood cell contamination than simple washed samples (0.02+/-0.01, 0.02+/-0.04, 0.03+/-0.04, and 0.44+/-0.22 x 10(6) cells/mL, respectively; P<0.05). Although the proportion of sperm with head abnormalities did not differ among selection methods (P>0.05), SLC-PC yielded the highest percentage of sperm with normal midpieces and tails (P<0.05), due to the lowest proportion of coiled tails (P<0.05). Furthermore, the SLC-PC was as effective as swim-up in removing sperm with proximal droplets, and selecting motile sperm, as well as those with intact membranes and DNA (P>0.05). In conclusion, both SLC-PC and swim-up improved the quality of epididymal cat sperm, including better morphology, membrane and DNA integrity, and removal of cellular contamination. However, SLC had a better sperm recovery rate than swim-up.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Effects of thawing temperature and post-thaw dilution on the quality of cat spermatozoa.

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70 degrees C) and to investigate the effects of post-thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25-ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37 degrees C for 15 s, (ii) at 37 degrees C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70 degrees C for 6 s, (iv) at 70 degrees C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR-14/EthD-1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 +/- 10.7 (mean +/- SD), a score of progressive motility of 4.0 +/- 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 +/- 12.1 and intact acrosome of 44.8 +/- 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70 degrees C and post-thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post-thaw incubation.

The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps)

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.

Detection of Lipid Peroxidation Reaction in Frozen-Thawed Epididymal Cat Spermatozoa Using BODIPY581/591 C11

Lipid peroxidation is considered to be an important cause of sperm membrane damage, resulting in an apparent reduction of reproductive fecundity. Recently, a new lipophilic fluorescent dye probe (BODIPY(581/591) C11; Invitrogen Singapore Pte Ltd, Singapore) has been demonstrated to be a highly sensitive indicator for the physiological oxidation of cell membrane fatty acids. The objectives of this study were: (i) to detect lipid peroxidation in frozen-thawed epididymal cat spermatozoa using the BODIPY(581/591) C11 and (ii) to study the effect of semen extender in protecting sperm membrane from lipid peroxidation [100-mm ferrous ion, ferrous sulphate (FeSO(4))]. Epididymal cat spermatozoa were collected from eight male cats. Two straws of sperm sample from each cat were cryopreserved. After thawing, the semen extender from the first straw was removed and the sperm pellet was resuspended with Tris buffer (control). The semen sample from the other straw was equally divided: one sample contained semen extender (treatment A) and one contained no extender (treatment B); both were incubated with FeSO(4). Semen samples were labelled with the BODIPY(581/591) C11 probe and evaluated by flow cytometry at 0 and 6 h after thawing (control), 6 h after the addition of FeSO(4) (treatment A), and 30 min and 6 h after the addition of FeSO(4) (treatment B), respectively. The percentage of lipid peroxidation was higher after treatment B (51.3 +/- 23.9) and 6-h incubation compared with the control and treatment A (p < 0.05). Furthermore, the percentage of lipid peroxidation after treatment B increased during the incubation time (p < 0.05). In conclusion, the high percentage of lipid peroxidation after treatment B indicated that FeSO(4) induced membrane damage in cat spermatozoa, which could be detected by BODIPY(581/591) C11. This marker is suggested to be a beneficial tool for the evaluation of lipid peroxidation. Furthermore, the use of semen extender seemed to protect cat spermatozoa membranes from lipid peroxidation.

Sperm quality and the morphology of cryopreserved testicular tissues recovered post-mortem from diverse wild species.

This study compared the effects of slow and fast freezing of testicular tissue of wild animals collected at post-mortem on testicular structure and testicular sperm. The testes of seven animals that had died in captivity; three felids (jungle cat, lion and leopard), two cervids (rusa deer and feas muntjac) and two bovids (Sumatran serows) were cryopreserved using slow- and fast-freezing protocols. There were greater reductions in the integrity of the sperm membrane and DNA in tissues cryopreserved using slow freezing compared to fast freezing (membrane integrity reduced by 21.5 ± 12.4% vs. 13.0 ± 6.9%, P = 0.11 and DNA integrity reduced by 22.7 ± 16.3% vs. 6.6 ± 6.3%, P = 0.13). Histologically, there were similar degrees of detachment and shrinkage of the seminiferous tubules whereas, TUNEL assay revealed a tendency towards more apoptotic changes in the intra-tubular cells of tissues frozen using fast freezing compared to slow freezing (P = 0.09). In conclusion, fast freezing tended to cause less damage to testicular sperm but its protective effect on intra-tubular cells was likely compromised. This is the first report of gamete recovery in the wild and of the comparison in various wildlife species, between testicular tissues cryopreserved using different protocols.

Cryopreservation of Cat Testicular Tissues: Effects of Storage Temperature, Freezing Protocols and Cryoprotective Agents

Cryopreservation of testicular tissue has become a part of gamete preservation in wild animal post-mortem. Using domestic cats as a model for wild felids, this study aimed to (i) investigate the effect of temperature for testicular tissue storage on sperm quality; (ii) compare efficiency of freezing protocols; and (iii) evaluate properties of cryoprotective agents to protect testicular sperm quality. A pair of testes from each cat (n = 9) was cut into four pieces. Three randomly selected pieces were allocated to be (i) fresh controls; (ii) stored at 4 °C for 24 h; and (iii) stored at room temperature (28 °C) for 24 h. After storage, the testicular tissue from each group was cut into 10 small pieces. One piece was assigned to be a control while the others were assigned to three freezing protocols; -80 °C (n = 3), vitrification (n = 3) or two-step freezing (kept above liquid nitrogen vapour for 10 min and submerged in liquid nitrogen) (n = 3). Each of three pieces was frozen using dimethyl sulphoxide (DMSO), ethylene glycol (EG) or DMSO combined with EG. Sperm membrane (SYBR-14/EthD-1) and DNA (acridine orange) integrity were evaluated before and after cryopreservation. The storage of testicular tissue at room temperature decreased the percentage of sperm with intact membrane in fresh tissue (59.5 ± 30.5 vs 87.9 ± 7.0%, p < 0.05). DNA integrity was decreased after 24-h storage either at 4 °C or room temperature (p < 0.05). The two-step freezing resulted in a higher percentage of sperm with intact plasma membrane than the other techniques. Dimethyl sulphoxide, EG and DMSO combined with EG provided similar protection for the sperm membrane and DNA from cryodamages. In conclusion, storage of testicular tissue at 4 °C is necessary to maintain sperm membrane integrity during transportation of tissue for cryopreservation in the freezing laboratory. The results provide information for male gamete rescue in felid particularly when they die unexpectedly in the field where freezing facilities are not well equipped.

Effects of Cold Storage Prior to Freezing on Superoxide Dismutase, Glutathione Peroxidase Activities, Level of Total Reactive Oxygen Species and Sperm Quality in Dogs

Reactive oxygen species (ROS) are one of several crucial factors that cause mammalian sperm damage during handling and preservation. Their deleterious effects can be restricted by the action of antioxidants. The present study aimed at investigating: (i) effects of cold storage prior to freezing on activities of enzymatic antioxidants (superoxide dismutase; SOD and glutathione peroxidase; GPx) and level of total reactive oxygen species (tROS); and (ii) effects of SOD or SOD plus GPx supplementation to chilled (3 and 96 h) and frozen-thawed semen. Six privately owned dogs were included. Experiment I: Each pooled semen was divided into three equal aliquots, which were subjected to chilled storage for 3, 24 or 96 h prior to freezing (n = 7). The activities of SOD and GPx in sperm cells and tROS level in chilled and frozen-thawed semen were measured. Experiment II: Pooled semen was divided to be cold stored for 3 or 96 h in three different extenders; (i) Uppsala Equex extender (control), (ii) Uppsala Equex extender supplemented SOD, or (iii) Uppsala Equex extender supplemented with SOD plus GPx. Sperm motility, viability and integrity of acrosome and DNA was evaluated after cold storage and frozen-thawed. The cold storage from 24 h prior to freezing resulted in a decrease in the SOD activity in the frozen-thawed sperm cells whereas the GPx activity and tROS levels were not affected. In addition, the supplementation of SOD plus GPx enhanced the percentage of sperm viability and DNA integrity after cold stored and frozen-thawed. In sum, the SOD activity is compromised by cold storage prior to freezing of dog semen. Addition of GPx is suggested to assist SOD to complete the enzymatic ROS scavenging system in the dog sperm.

Incubation of post-thaw epididymal cat spermatozoa with seminal plasma.

In domestic cats, epididymal spermatozoa have lower initial motility and viability than ejaculated spermatozoa and it is possible that seminal plasma compounds are behind these effects. The aim of this study was to investigate whether co-incubation of post-thaw epididymal cat spermatozoa with seminal plasma was able to improve sperm quality. Epididymal cat spermatozoa from 11 cats were cryopreserved. After thawing, each sperm sample was divided into two aliquots, centrifuged and incubated with two different media; Tris buffer (control) or pooled seminal plasma (treatment). Sperm quality was observed at 0, 2, 4 and 6 h after incubation. The results demonstrated that all of the sperm parameters except acrosome integrity were lower in the treatment group compared to the control group (p < 0.05); the percentages of motility (46.4 +/- 15.4 vs 40.0 +/- 9.4), the scores of progressive motility (3.1 +/- 0.4 vs 2.8 +/- 0.5), the percentages of spermatozoa with intact plasma membrane (46.3 +/- 9.7 vs 39.6 +/- 8.9) and intact acrosome (36.5 +/- 16.2 vs 32.9 +/- 15.1), as well as at all time points. In conclusion, the seminal plasma seems less beneficial to the post-thaw epididymal cat spermatozoa than the Tris buffer.