Economic opportunities of using crossbreeding and sexing in Holstein dairy herds.

With the increasing availability of sexed semen, farms have the opportunity to select genetically superior dams to produce their replacement animals and to produce crossbred calves for beef production of higher economic value than the remainder of the herd. However, higher costs and reduced fertility of sexed semen complicate the decision of when and to what extent sexed semen should be applied in a herd. The objective of this study was to explore the economically optimal utilization of sexed semen and crossbreeding among North Rhine-Westphalian dairy farms in a holistic single-farm model. For the analysis, we derived a representative sample of farms from Latin Hypercube sampling based on the observed distribution of farm characteristics from official North Rhine-Westphalian Farm Structure Survey data. Market- and technology-related input parameters such as output prices and sexed semen accuracy and fertility were included in the sampling procedure. Modeling results of the systematic sensitivity analysis were evaluated in a statistical meta-model. We found that the profit-maximizing utilization of sexed semen and crossbreeding was highly heterogeneous among the farms. Farms with lower stocking densities, <2 livestock units (LU)/ha, were generally found to produce excess heifers for sale, whereas farms with stocking densities >2 LU/ha were producing crossbred calves and using sexed semen only to produce replacement animals. On average, female-sexed dairy semen was used on 25.3% of all inseminations. Beef semen (both sexed and conventional) for producing crossbred calves was used in an average of 21.5% of the inseminations. The combination of sexed semen and crossbreeding increased profits from €0 to €568 per cow per year, with an average of €79.42 per cow per year. Farms characterized by low stocking densities (<2 LU/ha) and above-average replacement rates (>40%) were found to have higher profit increases as a result of selling more heifers from the use of sexed semen. Overall, sexed semen and crossbreeding adoption were most sensitive to stocking density and average cow longevity, as well as to additional costs for sexed semen and sexed semen accuracy. Our results show the potential of modern breeding technologies to improve dairy farm profits and the need to judge their profitability in the light of farm-specific production settings.

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

L-carnitine supplementation in culture media improves the pregnancy rate of in vitro produced embryos with sexed semen from Bos taurus indicus cows.

The in vitro embryo production industry in the actual world presents some difficulties related to low embryonic production rates, a problem that could be associated with in vitro culture conditions that differed from the in vivo (oviductal) conditions, mainly related to cytoplasmic lipid accumulation. L-carnitine is known as a modulator of ß-oxidation in the developing embryo, as it has been demonstrated that it improves embryo quality without affecting the in vitro embryo production rate. The aim of the present work was to evaluate the effect of L-carnitine supplemented during the in vitro maturation and culture processes on the implantation rate of in vitro produced embryos. Supplementation with 3.8 mM of L-carnitine was used during in vitro maturation, and later, during late in vitro culture, it was added at 1.5 mM. A control group contained no L-carnitine supplementation. Bovine oocytes obtained by ultrasound-guided follicle aspiration from healthy Bos taurus indicus cows were matured, fertilized and cultured in vitro. Multiparous F1 (Bos taurus taurus × Bos taurus indicus) cows were used as recipients. Overall, 460 oocytes were processed in three independent replicates from in vitro maturation until day 8 of the in vitro culture. No significant difference was found between treatments of in vitro embryo production. However, pregnancy rate at days 45 and 72 was significantly higher in blastocysts derived from L-carnitine treatment (31.55 ± 9.78%) compared to the control group (18.68 ± 6.31%). In conclusion, addition of L-carnitine at 3.8 mM and 1.5 mM in the maturation, and culture medium after day 3 of in vitro production process, significantly improved pregnancy rate after embryo transfer.

Deep insemination with sex-sorted Cashmere goat sperm processed in the presence of antioxidants.

Flow cytometrically sex-sorted sperm have been widely used for improving reproductive management in the dairy industry. However, the industrial application of this technology in other domestic species is largely limited by the lower fertility after insemination. The aim of this study was to investigate effects of antioxidant supplementation during the sex-sorting and freezing process on the quality and functions of sorted sperm from Liaoning Cashmere goats. We tested the effects of antioxidant supplementation during sex-sorting and freezing process, including ascorbic acid-2-glucoside AA-2G, glutathione, melatonin and vitamin C (VC), on the quality and functions of sex-sorted fresh and frozen-thawed sperm. Based on these experiments, we performed deep insemination with sex-sorted sperm using our improved strategy, in comparison to unsorted sperm. In Experiment 1, compared with control group and other antioxidants, AA-2G supplementation significantly alleviated the degradation of motility and viability of fresh sperm after sorting and showed the highest percentage of sperm with normal morphology. In addition, AA-2G supplementation showed an evident protection against the sorting process-induced membrane and acrosome damage. In Experiment 2, AA-2G supplementation was most effective in protecting motility, while melatonin supplementation appears to facilitate the degradation of quality of frozen-thawed sex-sorted sperm. In Experiment 3, we performed deep insemination with sperm that were sorted and frozen in the presence of AA-2G and obtained a satisfying pregnancy rate comparable to that from unsorted sperm. The results showed that AA-2G supplementation efficiently protects quality and function of both fresh and frozen-thawed sex-sorted sperm of Cashmere goats, thus obtaining a satisfying pregnancy outcome.

Quercetin improves the postthaw characteristics of cryopreserved sex-sorted and nonsorted stallion sperm.

Excessive reactive oxygen species generation during sex sorting and cryopreservation of stallion sperm leads to DNA fragmentation, lipid peroxidation, and motility loss. In this study we investigated whether antioxidant supplementation during sex sorting and cryopreservation could ameliorate the effects of reactive oxygen species on stallion sperm. In experiment 1, the postthaw characteristics of stallion sperm (N = 9) cryopreserved in the presence or absence of catalase (200 U/mL), cysteine (0.2 mg/mL), or quercetin (0.15 mM) was examined. Motility and acrosome integrity were assessed at 0, 1, and 3 hours after thawing. The sperm chromatin structure assay (SCSA; detectable DNA fragmentation index [DFI], mean DFI, and DFI) was used to assess DNA integrity immediately after thawing. Quercetin increased the total postthaw motility (25.3% vs. 20.9%; P < 0.05), but there was no beneficial effect of catalase or cysteine. Based on these results, the effect of quercetin during cryopreservation on the postthaw zona binding ability of sperm was assessed using a heterologous (bovine) zona binding assay. Quercetin increased the number of sperm bound per oocyte (13.6 vs. 9.2; P < 0.05) compared with the control. In experiment 2, the effect of quercetin (0.15 mM) in the media used during semen storage and transport, Hoechst 33342 staining and cryopreservation of stallion sperm (N = 9) was investigated. Motility, acrosome integrity, and viability were assessed at 0, 1, and 3 hours after thawing and SCSA was performed at 0 hours after thawing. Quercetin supplementation during sex sorting and cryopreservation improved DNA integrity (SCSA; detectable DFI of 54.9% vs. 74.6%, P < 0.05; mean DFI of 270.2 vs. 288.1, P < 0.05; and DFI of 26.3% vs. 28.5%, P < 0.05) compared with control sex-sorted sperm. There was no beneficial effect of quercetin on the motility, acrosome integrity, or viability of sex-sorted sperm. In conclusion, quercetin significantly improved the motility and zona binding ability of cryopreserved stallion sperm, and reduced DNA fragmentation in sex-sorted, cryopreserved stallion sperm.

Effect of antioxidant supplementation on function and fertility of sex-sorted boar spermatozoa.

The goal of this study was to investigate the effects of antioxidant supplementation on the quality of flow cytometrically-sorted boar spermatozoa. The effects of ascorbic acid-2-glucoside (AA-2G) on the sex-sorting process were evaluated using a variety of concentrations. The effects of different antioxidants (AA-2G, l-glutathione, and vitamin E) on the viability and lifespan of boar spermatozoa were also compared during sorting. Furthermore, the effect of AA-2G on acrosome intactness, the capacitation ability of sorted boar spermatozoa and pregnancy efficiency after artificial insemination (AI) at different sorting-to-insemination intervals were examined. Greater (P<0.05) percentages of motile spermatozoa and acrosome intactness and longer storage time periods were observed in the AA-2G-supplemented group when compared with the other antioxidant-supplemented or control groups. At an AA-2G concentration of 0.068 mg/mL, the motility characteristics (i.e., straightness (STR), velocity according to the average path (VAP), and amplitude of the sperm head lateral displacement (ALH)) of the sex-sorted boar spermatozoa were greater (P<0.05) than in those treated with other AA-2G concentrations. The capacitation rate of boar spermatozoa in the AA-2G-supplemented group was less (P<0.05) than that in the control group. After sorting-to-insemination interval of 10h, the pregnancy rates after AI with boar spermatozoa for the AA-2G-supplemented group were 59.25%, while the control group remains no sufficient quality semen. This study demonstrates that AA-2G supplementation can improve the quality of flow cytometrically sorted boar spermatozoa and that the optimal concentration of AA-2G for sorting is 0.068 mg/mL.

Protective effects of melatonin against oxidative stress in flow cytometry-sorted buffalo sperm.

Previous reports of the ability of melatonin to scavenge a variety of toxic oxygen and nitrogen-based reactants suggest that melatonin could be an effective antioxidant for protecting sperm. In this study, flow cytometry and laser tweezers Raman spectroscopy were used to evaluate the effect of melatonin on buffalo sperm quality to optimize sperm sex-sorting procedures. In fresh sperm incubated in the presence or absence of melatonin (10(-4) m) for 1, 24, 48 h or 72 h at 27°C, the mitochondrial activity was significantly higher than in a non-melatonin control (p < 0.05). Also, during the flow-sorting process, sperm in melatonin-supplemented groups had higher (p < 0.05) mitochondrial activity than the control. The intensity of Raman spectra from sperm frozen in media supplemented with melatonin was significantly weaker than that for non-melatonin-treated groups, except for a band at 1302 per cm. Thus, melatonin helps to protect buffalo sperm from reactive oxygen species induced by staining, sorting and freezing and increases semen quality after the freezing-thawing processes. Furthermore, the results indicate the high potential of the laser tweezers Raman spectroscopy technique for rapid, effective and non-invasive assessment of the quality of sperm cells.

The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa.

In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.