Development of Procedures for Sex-sorting Frozen–Thawed Bovine Spermatozoa

Dairy bull sperm may be sex‐sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well‐managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex‐sorting from frozen–thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex‐sort and re‐freeze frozen–thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm^® or BoviPure?, followed by staining in one of three diluents (Androhep^®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen–thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm^® rather than BoviPure? gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep^® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen–thawed bull sperm to be sex‐sorted with high resolution between the sexes, then re‐frozen and thawed with retention of motility and acrosome integrity.     

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post‐thaw semen characteristics

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen‐thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species‐specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell^® (IMV Technologies, l′Aigle, France); the SLC‐selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC‐treated samples had a higher percentage of live, superoxide‐positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non‐treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide‐positive spermatozoa and live, hydrogen peroxide‐negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC‐selected spermatozoa compared to unselected samples.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm

The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H₂O₂ 0 mm  = H000; H₂O₂ 50 mm  = H050; H₂O₂ 100 mm  = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H₂O₂ increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm‐sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non‐sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Does single-layer centrifugation with Bovicoll improve sperm quality of frozen-thawed semen in Fleckvieh bulls?

The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl^® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.     

Birth of Kids After Artificial Insemination with Sex‐Sorted,Frozen‐Thawed Goat Spermatozoa

Successful sex‐sorting of goat spermatozoa and subsequent birth of pre‐sexed kids have yet to be reported. As such, a series of experiments were conducted to develop protocols for sperm‐sorting (using a modified flow cytometer, MoFlo SX^®) and cryopreservation of goat spermatozoa. Saanen goat spermatozoa (n = 2 males) were (i) collected into Salamon's or Tris catch media post‐sorting and (ii) frozen in Tris–citrate–glucose media supplemented with 5, 10 or 20% egg yolk in (iii) 0.25 ml pellets on dry ice or 0.25 ml straws in a controlled‐rate freezer. Post‐sort and post‐thaw sperm quality were assessed by motility (CASA), viability and acrosome integrity (PI/FITC‐PNA). Sex‐sorted goat spermatozoa frozen in pellets displayed significantly higher post‐thaw motility and viability than spermatozoa frozen in straws. Catch media and differing egg yolk concentration had no effect on the sperm parameters tested. The in vitro and in vivo fertility of sex‐sorted goat spermatozoa produced with this optimum protocol were then tested by means of a heterologous ova binding assay and intrauterine artificial insemination of Saanen goat does, respectively. Sex‐sorted goat spermatozoa bound to sheep ova zona pellucidae in similar numbers (p > 0.05) to non‐sorted goat spermatozoa, non‐sorted ram spermatozoa and sex‐sorted ram spermatozoa. Following intrauterine artificial insemination with sex‐sorted spermatozoa, 38% (5/13) of does kidded with 83% (3/5) of kids being of the expected sex. Does inseminated with non‐sorted spermatozoa achieved a 50% (3/6) kidding rate and a sex ratio of 3 : 1 (F : M). This study demonstrates for the first time that goat spermatozoa can be sex‐sorted by flow cytometry, successfully frozen and used to produce pre‐sexed kids.     

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield,motility and survival

Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Nitric oxide impacts bovine sperm capacitation in a cGMP‐dependent and cGMP‐independent manner

We aimed to elucidate whether NO acts in in vitro sperm capacitation in bovine via cGMP/PKG1 pathway. For this, cryopreserved bovine sperm were capacitated in vitro with 20 µg/ml heparin (Control) plus treatments: 1 mM L‐arginine (L‐arg, NO precursor), 50 µM Rp‐8‐Bromo‐β‐phenyl‐1,N²‐ethenoguanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐Br‐cGMPS, selective inhibitor of the binding site for cGMP in PKG1), 1 mM 2‐Phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide (PTIO, NO scavenger), and the combinations of L‐arg + RP‐8‐Br‐cGMPS and L‐arg + PTIO. Sperm motility and vigour were determined by phase‐contrast microscopy, capacitation status by chlortetracycline staining, and the intracellular concentration of cGMP was measured by ELISA. Data were subjected to analysis of variance and means compared with SNK test at 5% probability. Motility and vigour were lower in sperm treated with PTIO when compared to Control and other treatments (p < .05). The L‐arg treatment showed the highest percentage of capacitated sperm when compared to the Control and other treatments (Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO) (69.8 ± 3.4%, 51.2 ± 3.0, 51.1 ± 2.1, 51.2 ± 3.0 and 45.5 ± 2.7, respectively) (p < .05). The capacitation ratio (%) was lower in treatments with Rp‐8‐Br‐cGMPS, L‐arg + Rp‐8‐Br‐cGMPS and PTIO, respectively (p < .05). Lastly, cGMP concentration (pmol/ml) was lower in PTIO and L‐arg + PTIO (1.3 ± 0.3 and 1.6 ± 0.4) and was higher in Rp‐8‐Br‐cGMPS and L‐arg + Rp‐8‐Br‐cGMPS (3.7 ± 0.4 and 4.0 ± 0.5) treatments. We showed that during in vitro capacitation of cattle: (a) NO influences sperm motility and vigour; (b) NO is associated with cGMP synthesis through two independent pathways and (c) the cGMP/PKG1 pathway has a partial role in sperm capacitation and does not involve the L‐arg/NO.     

Superovulation protocols for dairy cows bred with SexedULTRA™ sex‐sorted semen

The objective was to compare embryo yield and quality in lactating dairy cows superovulated (SO) with varying amounts of gonadotropins and FSH:LH ratios and inseminated with SexedULTRA? sex‐sorted semen. The SO treatments (n = 77) involved 3 protocols: groups F700 and F1000 were given total doses of 700 and 1,000 IU of Folltropin (FSH:LH ratio 49:1), respectively, whereas group F700P300 was given 700 IU of Folltropin + 300 IU of Pluset (FSH:LH ratio 1:1). Cows were artificially inseminated 3 times over a 10‐hr interval with frozen‐thawed SexedULTRA? sex‐sorted semen (total of 10 × 10⁶ sex‐sorted sperm), starting 18 hr after onset of oestrus, with embryos/ova recovered 7 d after oestrus. Total number of recovered structures and transferable embryos were lower (p < 0.05) in F700 (4.7 ± 3.0 and 1.9 ± 1.7, respectively; mean ± SD) compared to F1000 (8.1 ± 3.8 and 4.4 ± 2.6) and F700P300 (8.5 ± 6.4 and 4.5 ± 3.3). Percentage of cows ovulating >50% of follicles ≥0.8 cm in diameter was lower (p < 0.05) in F700 (35.5%) than in F1000 (82.4%) and F700P300 (73.1%). Percentage of unfertilized oocytes was higher (p < 0.05) in F700 (45.0% vs. 27.7% for F1000 and 29.0% for F700P300) whereas percentage of morulae was higher (p < 0.05) in F1000 (19.3% vs. 8.7% for F700 and 12.2% for F700P300). Embryo quality was similar among groups (p > 0.05). In conclusion, embryo production in lactating dairy cows was improved by increasing total dose of gonadotropins from 700 to 1,000 IU, with SexedULTRA? sex‐sorted semen yielding satisfactory fertilization rates and embryo quality.     

Single Layer Centrifugation with 20% or 30% Porcicoll separates the majority of spermatozoa from a sample without adversely affecting sperm quality

Centrifugation of boar semen through one layer of 40% colloid (Porcicoll) was previously shown to separate spermatozoa from bacteria without having a detrimental effect on sperm quality. However, some spermatozoa were lost. The purpose of the present study was to determine whether 20% or 30% Porcicoll could be used to recover most of the spermatozoa without impacting on sperm quality. Insemination doses (n = 10) from a commercial boar station were sent to the laboratory at the Swedish University of Agricultural Sciences and processed by Single Layer Centrifugation with 20% and 30% Porcicoll approximately 7 hr after semen collection. The resulting sperm samples and controls were evaluated for sperm quality immediately and again after storage at 16–18°C for 4 and 7 days. Sperm recovery was 94 ± 18% and 87 ± 15% for 20% and 30% Porcicoll, respectively (p > .05). Sperm mitochondrial membrane potential and chromatin integrity were unaffected (p > .05). The proportion of live spermatozoa producing superoxide (9 ± 8%, 7 ± 6% and 3 ± 1%; p < .05), and the proportion of spermatozoa with high stainability DNA (0.68 ± 19%, 0.61 ± 0.22% and 0.96 ± 0.23%; p < .05- <0.01), were marginally increased whereas membrane integrity, although high, was lower in the centrifuged samples than in the controls (82 ± 8%, 83 ± 5% versus 92 ± 4%; p < .05). In conclusion, centrifugation through 20% or 30% Porcicoll enables most spermatozoa to be recovered, without having a major effect on sperm quality. These results are encouraging for further studies involving microbiological investigation of the processed samples, and scaling-up to process larger volumes of boar ejaculates.     

Fertility in Single‐ovulating and Superovulated Dairy Heifers after Insemination with Low Dose Sex‐sorted Sperm

The present study was performed to test fertility in single‐ovulating and superovulated dairy heifers after insemination with low dose sex‐sorted sperm under field conditions. Some parameters, including the dosage, deposition site and timing, were assessed with the pregnancy rates after artificial insemination (AI). Moreover, the use of oestrus synchronization in combination with sorted sperm was evaluated. Besides that, we also improved the embryo production efficiency in superovulated dairy heifers by optimizing the timing of inseminations and repartitioning the sexed sperm dosage among multiple inseminations. The conception rate (52.8%) in heifers after low dose (2 × 10⁶) insemination with sorted sperm deep into the uterine horn did not differ (p > 0.05) from that (59.6%) of conventional AI (1 × 10⁷ non‐sorted sperm) and that of deep insemination with low dose non‐sorted sperm (57.7%). There was also no difference (p > 0.05) between conception rates after single (51.7%) and double (53.8%) deep insemination with sorted semen. Heifers inseminated with sorted sperm at synchronous oestrus had a lower pregnancy rate (48.1%) than heifers at spontaneous oestrus (53.6%), but this did not reach statistical difference (p > 0.05). The average number of transferable embryos collected in vivo from heifers inseminated with sorted sperm (4.81 ± 2.04) did not differ (p > 0.05) from that obtained from heifers after insemination with non‐sorted sperm (5.36 ± 2.74). Thus, we concluded that the pregnancy rate after deep intra‐uterine insemination with low dose sorted sperm was similar to that of non‐sorted sperm, which was either also deposited at a low dose deep intra‐uterine or into the uterine body. Sychronization of oestrus can be beneficial in combination with sorted sperm to optimize the organization and management of dairy herds. The results from superovulated heifers demonstrated that our insemination regime can be used to obtain a comparable embryo production efficiency with sorted sperm than with non‐sorted sperm.     

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted,frozen–thawed spermatozoa in field conditions

The aim of this study was to evaluate fertility and sex ratios after artificial insemination in dogs under field conditions. Semen was cryopreserved as unsorted (control) or was separated into X‐ and Y‐chromosome‐bearing sperm using a cell sorter. Sixty female dogs were inseminated with frozen–thawed spermatozoa of 100 × 10⁶ unsorted (a dose in practice) and 4 × 10⁶ sorted (X and Y group, respectively). A total of 20 dogs became pregnant and 126 puppies were born from the three groups. The percentage of parturition was similar for the X (5/20; 25.0%) and Y (4/20; 20.0%) group (P > 0.05), but lower than controls (11/20; 55.0%) (P < 0.05). Ultimately 28 out of the 32 puppies produced from X group were female (87.5%) and 19/22 (86.4%) puppies of Y group were male. In contrast, sex ratio (51.4% to 48.6%) in the control was significantly different from the X, Y group (P < 0.05). However, male and female puppies in the control had similar birth weights and weaning weights to those from the X and Y groups. This preliminary information indicated that normal puppies of predicted sex can be produced with low numbers of sorted cryopreserved dog spermatozoa at a farm level, making sperm‐sexing technology potentially applicable for elite breeding units.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

Silica-based colloid centrifugation enhances sperm quality in cockerel semen

ABSTRACT

1. Percoll^TM is one of the most widely used colloid for animal sperm preparation. The aim of this study was to evaluate whether Percoll^TM colloid centrifugation could be practical to improve cockerel sperm quality, and to compare the effects of Percoll^TM single layer centrifugation (SLC) and density gradient centrifugation (DGC) in order to obtain the most optimal protocol for cockerel semen.

2. In the experiment with Percoll^TM SLC for fresh semen, an increase of motile sperm was seen after Percoll^TM 80% SLC and 90% SLC was conducted, at levels of 28.8% and 30.2% respectively (P < 0.01). The increase of progressively motile sperm after Percoll^TM 80% SLC and 90% SLC was 177.2% and 202.4% respectively (P < 0.01). Meanwhile, for semen stored at 4°C for 24 h, the increase of motile sperm after Percoll^TM 70% SLC and 80% SLC was 41.2% and 44.0% (P < 0.01), and the increase of progressive sperm after Percoll^TM 70% SLC and 80% SLC was 71.3% and 83.1% respectively (P < 0.01). Both the percentage of motile sperm and progressive sperm of the fresh and stored cockerel semen after appropriate Percoll^TM SLC was significantly enhanced.

3. Sperm membrane integrity did not show any decrease after Percoll^TM centrifugation compared with non-centrifuged semen, which suggested that the Percoll^TM centrifugation treatment in this study did not cause damage to cockerel sperm membranes.

4. In the experiment regarding the comparison of Percoll^TM SLC and DGC with fresh semen, the increase of motile sperm after Percoll^TM 80% SLC, 90% SLC and 40%/80% DGC was 29.5%, 36.4%, and 25.0% respectively; and the increase of progressive sperm was 44.7%, 58.5%, and 54.7%, respectively. For semen stored at 4°C for 24 h, the increase of motile sperm after Percoll^TM 70% SLC, 80% SLC and 35%/70% DGC were 41.2%, 44.0%, and 26.4%; and the increase of progressive sperm was 71.3%, 83.1%, and 43.7%, respectively. There were no significant differences between the increase of sperm motility after Percoll^TM 80%, 90% SLC or Percoll^TM 40%/80% DGC in fresh cockerel semen. There was no significant difference between Percoll^TM 70%, 80% SLC and Percoll^TM 35%/70% in stored cockerel semen. There was a tendency for sperm recovery rates with Percoll^TM SLC to be higher than Percoll^TM DGC, although this did not reach statistical significance in this study.

5. It was concluded that Percoll^TM SLC was more suitable for cockerel sperm separation than Percoll^TM DGC. The results suggested that Percoll^TM 80% SLC was the most optimal procedure to separate fresh cockerel sperm and Percoll^TM 70% SLC was the most optimal procedure to separate stored cockerel sperm. Percoll^TM SLC is more simple, user-friendly and economical and less time-consuming than DGC for cockerel semen processing.     

Evaluation of a portable device for assessment of motility in stallion semen

In horse breeding, quality assessment of semen before insemination is often requested. Non‐laboratory‐based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer‐assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 10⁶ sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 10⁶ sperm/ml when compared to 25 × 10⁶ sperm/ml (p < 0.05) but not when compared to 50 × 10⁶ sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland–Altman plot. Intra‐assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 10⁶ sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 10⁶ sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 10⁶ sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably.     

Comparative evaluation of DNA integrity using sperm chromatin structure assay and Sperm‐Ovis‐Halomax during in vitro capacitation of cryopreserved ram spermatozoa

This study aimed to compare the ability of sperm chromatin structure assay (SCSA^®) and Sperm‐Ovis‐Halomax^® to detect DNA fragmentation in frozen‐thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF‐ESS) at multiple time points (0–240 min). Incubation in SOF‐ESS had no significant effects on SCSA^® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm‐Ovis‐Halomax^® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA^® and Sperm‐Ovis‐Halomax^® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.     

Red‐light stimulation of boar semen prior to artificial insemination improves field fertility in farms: A worldwide survey

A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED‐based red light increases its reproductive performance following artificial insemination (AI). Red‐light stimulation with MaXipig^® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red‐light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red‐light stimulation. Our results indicate that a routine use of red‐light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.