Large volume cryoprotectant‐free vitrification: an alternative to conventional cryopreservation for human spermatozoa

Vitrification is a simple and cost‐effective method for the storage of human spermatozoa without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen. As a result, solidification of living cells without the formation of ice crystals is achieved during cooling. This study aimed to compare cryoprotectant‐free vitrification to conventional cryopreservation protocols. Semen samples (n = 35) were collected from patients seeking diagnostic assistance at the Reproductive and Endocrine Unit at Steve Biko Academic Hospital. Samples were processed using a discontinuous density‐gradient centrifugation method. Washed samples were split into two aliquots and cryopreserved either by means of cryoprotectant‐free vitrification (sucrose + 1% albumin) or conventional slow freezing (TEST‐yolk buffer). Post‐thawing, the sperm motion parameters, mitochondrial membrane potential (Δψm) and DNA fragmentation were compared between the two groups. No significant differences were observed in the sperm motility parameters (P > 0.05). Significantly higher percentages of Δψm (11.99% ± 4.326% versus 6.58% ± 1.026%; P < 0.001) and lower percentages of DNA fragmentation (2.79% ± 1.017% versus 3.86% ± 1.38%; P < 0.01) were observed when comparing cryoprotectant‐free vitrification to conventional cryopreservation. Cryoprotectant‐free vitrification is a rapid and promising alternative to conventional methods resulting in good‐quality spermatozoa post‐thaw.     

Giant panda (Ailuropoda melanoleuca) sperm morphometry and function after repeated freezing and thawing

This work examines the effects of subsequent cycles of freezing–thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density‐gradient centrifugation (DGC) can increase the number of freezing–thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing–thawing cycles. Although repeated freezing–thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG‐selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 μm, the head width 3.6 μm, area 14.3 μm² and perimeter length 14.1 μm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing–thawing, even without DGC selection.     

Relationship between donor sperm parameters and pregnancy outcome after intrauterine insemination: analysis of 2821 cycles in 1355 couples

The aim of this study was to investigate whether sperm parameters can affect the pregnancy outcome of artificial intrauterine insemination with cryopreserved donor spermatozoon (AID). A total of 1355 couples received 2821 AID treatment cycles in the Reproductive Medicine Center of the Tongji Medical College between January 2010 and December 2013, and the data were collected and retrospectively analysed. The relationship between pre‐freezing, post‐thawing as well as optimised sperm parameters and AID pregnancy outcome was investigated. Clinical pregnancy rate and cumulated pregnancy rate were also calculated. A total of 728 cycles from 2821 treatment cycles achieved pregnancies, and cumulated pregnancy rate was 25.81%. Pre‐freezing progressive sperm motility in pregnant cycles was higher than that in nonpregnant cycles (P = 0.001); logistic regression analysis also indicated that pre‐freezing progressive sperm motility was the only parameter affecting pregnancy outcome (P = 0.0001). Our study also showed that the cumulated pregnancy rate increased progressively and reached a plateau after the fifth cycle. In conclusion, pre‐freezing progressive sperm motility should be a valuable predictor for AID pregnancy outcome. Female fertility factors should be considered, or IVF/ICSI should be recommended when couples received more than 5 AID cycles without pregnancy.     

Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa

The aim of this study was to determine the effect of two equilibration temperatures (5 °C and room temperature) and two cryoprotectants (glycerol and dimethylformamide, both at 7%) on llama sperm cryopreservation. Llama ejaculates were divided into four aliquots. A lactose‐EDTA‐egg yolk (LEEY) extender with either 7% glycerol (LEEY‐G) or 7% dimethylformamide (LEEY‐DMF) was added to two of the aliquots, which were equilibrated for 20 min at room temperature and subsequently frozen. The other two aliquots were extended in LEEY, cooled to 5 °C, then LEEY‐G or LEEY‐DMF was added, equilibrated for 20 min at 5 °C and frozen. No significant differences (P > 0.05) were observed in membrane function and chromatin condensation between any of the freeze–thawing protocols. Post‐thaw motility was greater (P < 0.05) in LEEY‐DMF than LEEY‐G. DNA fragmentation was not different between raw and frozen semen with LEEY‐DMF but was high in all samples with glycerol. Our results indicate that 7% glycerol would be detrimental for llama spermatozoa, but further studies are needed to evaluate effectiveness if used at lower concentrations. Dimethylformamide preserved motility and DNA integrity of frozen–thawed llama spermatozoa and could be used to replace glycerol at the concentrations used in this study.     

Antioxidant effect of lemon balm (Melissa officinalis) and mate tea (Ilex paraguensys) on quality,lipid peroxidation and DNA oxidation of cryopreserved boar epididymal spermatozoa

In this study, we investigated the protective ability of the addition of two antioxidant herb extracts, mate tea and lemon balm, on boar epididymal frozen–thawed spermatozoa quality. Testes from mature boars were collected at local slaughterhouse, and sperm samples from epididymis were recovered by flushing. Spermatozoa were cryopreserved in lactose–egg yolk buffer supplemented with various concentrations of lemon balm and mate tea (0, 2.5, 5 and 10 g l^?1) using the straw‐freezing procedure. Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels and DNA oxidative damage (8‐hydroxy‐2′‐deoxyguanosine base lesion) were evaluated. There were no differences among experimental groups with regard to motility characteristics, viability, acrosome and plasma membrane integrity; however, the highest concentration of lemon balm produced significant (P < 0.05) improvement in curvilinear trajectory, straightness and amplitude of lateral head displacement after thawing. The supplementation of freezing extender with mate tea and lemon balm reduced sperm lipid membrane peroxidation, and only mate tea protected DNA against oxidative damage during cryopreservation at 120 min post‐thawing (P < 0.05). Mate tea experimental extender at concentration of 10 g l^?1 showed the lowest percentage of sperm oxidised DNA and malondialdehyde generation; thus, mate tea is a potential candidate such as antioxidant compound on boar sperm cryopreservation medium.     

The presence of seminal plasma,especially derived from stallion semen,helps preserve chilled Asian elephant (Elephas maximus) sperm motility

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris–glucose–egg yolk extender, supplemented with different SP types and preserved at 4°C for 48 hr. Experiment 1 (n = 31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p < .05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p < .05). Experiment 2 (n = 14) determined the effect of different SP from four stallions. All stallion SP gave higher (p < .05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage.     

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze‐thawing.     

Cryoprotectant‐free ultra‐rapid freezing of human spermatozoa in cryogenic vials

The objective of this study was to investigate the effect of ultra‐rapid freezing (direct immersion in liquid nitrogen) on human spermatozoa in cryogenic vials (≥0.5 ml) at different concentrations of sucrose. After swim‐up, the sperm suspensions (N = 58) were diluted with sperm preparation medium and divided into six aliquots: swim‐up (fresh), conventional freezing group (slow freezing) and four ultra‐rapid freezing groups containing sucrose at different concentrations (0.15 m , 0.20 m , 0.25 m and 0.30 m ). Sperm motility, progressive motility, plasma membrane integrity, DNA stability and acrosome integrity of fresh and cooled‐warmed spermatozoa were analysed. The progressive motility, plasma membrane and acrosome integrity of spermatozoa in the 0.20 m sucrose group were significantly higher than those of the slow freezing group (47.5 ± 6.8% versus 36.4 ± 8.7%, 73.2 ± 6.9% versus 63.9 ± 6.3%, 53.7 ± 10.0% versus 35.9 ± 9.7% respectively, P < 0.05). However, no differences were found in sperm motility or DNA stability (58.5 ± 6.3% versus 54.2 ± 5.3%, 90.1 ± 2.8% versus 87.2 ± 4.7%, P > 0.05 respectively) between the 0.20 m sucrose and the slow freezing group. No differences were found between the ultra‐rapid and slow freezing group at the other concentrations of sucrose. Our findings suggest that the method of ultra‐rapid freezing of human spermatozoa in cryogenic vials with a solution containing 0.20 m sucrose results in recovery of spermatozoon of superior qualities. In contrast to slow freezing, the ultra‐rapid freezing technique of human spermatozoa seems to reduce cryoinjuries and maintain important physiological characteristics of the spermatozoa after warming.     

Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification

This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre‐freezing equilibration times (2–3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm ). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60–85°C min^?1), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm ) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified–warmed sperm variables were at their best when the spermatozoa was diluted in TCG–6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.     

The influence of cryoprotective media and processing procedures on motility and migration of frozen—thawed human sperm

AIM: The study was designed to examine the effects of cryoprotective media, and glycerolating and thawing procedures on human sperm motility and gel penetrating ability. METHOD: Fifteen unselected donors provided semen varying in quality that was distributed in a factorial design across three cryoprotectants (glycerol, egg yolk-citrate-glucose-glycerol and egg yolk-tris-glucose-glycerol). Also, glycerol was added at room temperature versus at 4 degrees C. Two thaw temperatures were tested (laboratory air temperature for 10 min versus a 65 degrees C waterbath for 4 seconds). The proportion of total and progressively motile sperm was estimated immediately after thawing and following incubation at 35 degrees C for 2 h. Migration of sperm for 30 min at 37 degrees C through polyacrylamide gel was tested.RESULTS: Donors differed greatly, with post-thaw total motility of sperm ranging from 9 to 44% (P<0.05). Egg yolk-citrate-glucose-glycerol and egg yolk-tris-glucose-glycerol were superior to glycerol alone (post-thaw values of 35, 37 and 21%, respectively, P<0.05). This was due primarily to poor sperm survival when semen was cooled to 4 degrees C without glycerol or egg yolk. The two thaw temperatures gave similar results. Sperm migration tests paralleled the motility results, but were more sensitive in detecting differences. CONCLUSION: Egg yolk, particularly in a tris-based medium that is widely used in domestic animals, improved the cryopreservation of both good and poor quality human semen.     

Comparison of Tris egg yolk‐based,Triladyl® and Optixell® extender on post‐thaw quality,Kinematics and in vivo fertility of Nili Ravi Buffalo (Bubalus bubalis) bull spermatozoa

Our objectives were to ascertain the comparison of Tris egg yolk‐based, Triladyl ^® and Optixell ^® extender on postthaw quality, CASA parameters and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Semen samples (n = 35) from five bulls were diluted in Tris egg yolk‐based, Triladyl ^® , Optixell ^® extender and frozen in 0.50 ml French straws. Postthaw sperm CASA motility (%) was higher (p < 0.05) in Optixell extender as compared to Triladyl and Tris egg yolk‐based extender. Although sperm progressive motility (%), morphology (%), average path velocity (μm/s), straight line velocity (μm/s), curvilinear velocity (μm/s), amplitude of lateral head displacement (μm), beat cross‐frequency (Hz), straightness (%), length of curvilinear path (μm), length of average path (μm), intact plasma and acrosome membrane (%), and DNA integrity (%) were higher (p < 0.05) in spermatozoa cryopreserved in Optixell ^® extender as compared to Tris egg yolk‐based and Triladyl ^® extender. The fertility rates (68.18%, 45.45%, 55.4%) were higher (p < 0.05) in buffaloes inseminated with semen doses frozen in Optixell extender than the Tris egg yolk‐based and Triladyl ^® extender respectively. It is concluded that Optixell ^® extender improves postthaw semen quality and fertility in Nili Ravi buffaloes.     

A comparative study of two cooling protocols on stallion sperm cryosurvival

There are many protocols for horse sperm cryopreservation, but results are inconsistent; sperm survival after freeze‐thawing is usually poor; in consequence, fertility is low. The objective of this work was to see whether slow cooling before freezing to minus 3 °C instead of +5 °C, the traditional target temperature, could improve horse sperm cryosurvival, capability to carry out capacitation and the acrosome reaction induced by progesterone. Spermatozoa from five stallions were packaged in straws and slowly cooled to +5 °C. Half of the straws were frozen directly and the other half was further cooled to ?3 °C before freezing. Progressive motility, viability, plasma membrane integrity, acrosome integrity and capacitation status were assessed. After thawing, there were no differences between cooling treatments on motility, viability, acrosome integrity and capacitation status; however, there was difference (P < 0.05) regarding plasma membrane integrity. Acrosome integrity decreased as incubation, without or with progesterone (2 μg ml^?1), progressed, but there were no differences between cooling treatments regardless of progesterone. Both capacitated and acrosome‐reacted spermatozoa increased as incubation progressed, but there were no differences between cooling treatments regardless of progesterone. Slow cooling to ?3 °C before freezing did not improve horse sperm cryosurvival or capability to undergo the acrosome reaction.     

Cryoprotectant effect of trehalose in extender on post‐thaw quality and in vivo fertility of water buffalo (Bubalus bubalis) bull spermatozoa

This study was designed to ascertain the cryoprotectant effects of different concentrations of trehalose [0 (T0), 25 (T25), 35 (T35), 45 (T45) mm ], egg yolk [20% (E20), 15% (E15) v/v] and glycerol [7% (G7), 5% (G5) v/v] in Tris‐citric acid‐based extender on post‐thaw quality and in vivo fertility of buffalo bull spermatozoa. Twenty‐five ejaculates were collected from five bulls and split into four parts. After that, the split ejaculates from each of the bull were diluted either in T0E20G7 (control) or T25E20G5 or T35E15G5 or T45E15G5 extender. Finally, the sperm suspension was frozen in 0.54‐ml French straws. Post‐thaw sperm total motility (%), progressive motility (%), rapid velocity (%), average path velocity (μm/s), straightline velocity (μm/s), curvilinear velocity (μm/s), linearity (%), plasma membrane and acrosome integrities (%) were higher (p < .05) in T45E15G5 extender as compared to other treatment groups and control. The fertility rate (56.8% versus 41.3%) was higher (p < .05) in buffaloes inseminated with semen doses cryopreserved in extender containing T45E15G5 combination of cryoprotectants than the control. In conclusion, addition of 45 mm trehalose along with 15% egg yolk and 5% glycerol in extender improves the post‐thaw quality and in vivo fertility of buffalo bull spermatozoa.     

Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing

In this study, we compared the effects of repeated freezing/thawing of human sperm by our in-house method of rapid freezing with slow programmable freezing. Sperm samples from 11 normozoospermic subjects were processed through density gradients and divided into three aliquots: non-frozen, rapid freezing and slow programmable freezing. Sperm in the rapid freezing group had better motility and viability than those in the slow freezing group (P<0.01) after the first, second and third cycles of freezing/thawing, but there was no difference in morphology. In the second experiment, rapid freezing was repeated three times in 20 subjects. The samples from each thawing cycle were evaluated for DNA fragmentation using the alkaline comet assay. DNA fragmentation began to increase considerably after the second cycle of freezing/thawing, but to a level that was not clinically important. In the third experiment, rapid freezing was done repeatedly in 10 subjects, until no motile sperm were observed after thawing. The median number of repeated freezing/thawing that yielded no motile sperm was seven (range: 5–8, mean: 6.8). In conclusion, we demonstrated that repeated freezing/thawing of processed semen using our rapid freezing method gave better results than standard slow programmable freezing. This method can help maximize the usage of precious cryopreserved sperm samples in assisted reproduction technology.     

Testicular spermatozoon is superior to ejaculated spermatozoon for intracytoplasmic sperm injection to achieve pregnancy in infertile males with high sperm DNA damage

The purpose of this study was to compare the clinical outcome of testicular spermatozoon versus ejaculated spermatozoon in the treatment of infertile males with high sperm DNA damage, referred as sperm DNA fragmentation index (DFI), that attending intracytoplasmic sperm injection (ICSI) programme in terms of clinical pregnancy, births delivered as the primary and pregnancy loss and embryo fertilisation as the secondary outcome. A total of 102 males fulfilling the inclusion criteria were enrolled in the present study. Of the 102 males, 61 infertile males underwent testicular spermatozoon combined with ICSI while the remaining 41 males applied ejaculated spermatozoa in their first ICSI cycles, and the data of them were collected and analysed. In a 18‐month follow‐up, testicular spermatozoon achieved higher pregnancy rate and deliver rate than those in ejaculated sperm group (pregnancy rate, 36% vs. 14.6%, p = 0.017; deliver rate, 38.5% vs. 9.8%, p = 0.001). Nevertheless, there were no significant differences in the number of oocytes aspirated and number of embryos transferred between the two groups. Additionally, the fertilisation rate in the testicular sperm study cohort (70.4%) was also similar to that in the ejaculated sperm group (75.0%). Based on the current data, we conclude that testicular spermatozoon is the prior option in the treatment of infertile males with high sperm DFI in ICSI programme. More high‐quality studies with larger samples size are needed in the future due to the relative small size and the nonrandomized design of the present study.     

Effects of caffeine supplementation in post‐thaw human semen over different incubation periods

This study aimed to evaluate the effects of caffeine supplementation in post‐cryopreservation human semen over different incubation periods. After collection by masturbation, 17 semen samples were analysed according to World Health Organization criteria, processed and cryopreserved with TEST‐yolk buffer (1 : 1) in liquid nitrogen. After a thawing protocol, samples were incubated with 2 mm of caffeine for 0, 5, 15, 30 or 60 min, followed by analysis of motility and mitochondrial activity using 3,3′‐diaminobenzidine (DAB). Mean variance analysis was performed, and P < 0.05 was the adopted significance threshold. Samples incubated for 15 min showed increased progressive motility compared to other periods of incubation, as well as a reduced percentage of immotile spermatozoa (P < 0.05). In samples incubated for 5 min, increased mitochondrial activity above 50% was observed (DABI and DABII). Although cryosurvival rates were low after the cryopreservation process, incubation with caffeine was associated with an increase in sperm motility, particularly 15‐min incubation, suggesting that incubation with caffeine can be an important tool in patients with worsening seminal quality undergoing infertility treatment.     

Can SCSA and TUNEL forecast apoptosis‐related motility depletion in Asthenozoospermia?

This study is an attempt to determine the power of SCSA and TUNEL for the evaluation of apoptosis status and apoptosis‐related motility depletion in Asthenozoospermia. Fifty‐one semen samples from Asthenozoospermic and 20 samples from fertile men participated in this study. SCSA and TUNEL were applied for the assessment of DNA integrity by flow cytometry. Annexin V conjugated with FITC labelling and FLICA method were used for the assessment of externalisation of phosphatidylserine and spermatozoon with active Caspase 3 respectively. SCSA results were shown to have a significant correlation with EPS in live spermatozoon (r = .85, p value = .00) and spermatozoon with active Caspase 3 (r = .633, p value = .00). TUNEL result was revealed to have a nonsignificant positive correlation with them. Then, Asthenozoospermic individuals were divided into two groups, SCSA higher and SCSA lower than 27%. Results interestingly indicated that the two groups significantly differed from each other in terms of TUNEL, EPS in live spermatozoon, spermatozoon with active Caspase 3 and sperm vitality (p value = .00). Both SCSA and TUNEL were correlated with apoptosis‐related motility depletion in Asthenozoospermia. However, SCSA might be more powerful than TUNEL and could provide reliable information about DNA, chromatin integrity and apoptosis status in Asthenozoospermia.     

Effect of cysteine and glutamine added to extender on post‐thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris‐egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post‐thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen–thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen–thawed spermatozoa.     

Evaluation of the cryoprotective effect of seminal plasma on llama (Lama glama) spermatozoa

In South American camelids, sperm survival is low after thawing and poor results are obtained when artificial insemination is performed with cryopreserved semen. The aim of this study was to evaluate the effect of different percentages (10% and 50%) of seminal plasma added prior to the process of cryopreservation and also to evaluate the absence of seminal plasma on llama sperm survival after freezing and thawing. A total of 15 ejaculates from five adult llama males (n = 5; r = 3) were evaluated. A significant decrease in sperm motility, viability, membrane function and intact acrosomes was observed in thawed samples (0%, 10% and 50%) when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > 0.05), but a significant increase (p < 0.05) in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples compared to raw semen. Higher percentages of total and progressive sperm motility were observed when 0% and 10% of seminal plasma were used compared to 50%. However, no statistical differences were established for sperm viability, membrane function, morphology, acrosome status and DNA quality between thawed treatments. To conclude, neither of the percentages of seminal plasma used showed superiority nor cryoprotective effect on llama sperm survival.     

Utilisation of sperm‐binding assay combined with computer‐assisted sperm analysis to evaluate frozen‐thawed bull semen

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm² of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r² = 0.91 and CASA: r² = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.