Development of an index that decreases birth weight, promotes postnatal growth and yet minimizes selection intensity in beef cattle.

OBJECTIVE: The main goal of our current study was to improve the growth curve of meat animals by decreasing the birth weight while achieving a finishing weight that is the same as that before selection but at younger age. METHODS: Random regression model was developed to derive various selection indices to achieve desired gains in body weight at target time points throughout the fattening process. We considered absolute and proportional gains at specific ages (in weeks) and for various stages (i.e., early, middle, late) during the fattening process. RESULTS: The point gain index was particularly easy to use because breeders can assign a specific age (in weeks) as a time point and model either the actual weight gain desired or a scaled percentage gain in body weight. CONCLUSION: The point gain index we developed can achieve the desired weight gain at any given postnatal week of the growing process and is an easy-to-use and practical option for improving the growth curve.

Genomic dissection of repeatability considering additive and nonadditive genetic effects for semen production traits in beef and dairy bulls.

The low heritability and moderate repeatability of semen production traits in beef and dairy bulls suggest that nonadditive genetic effects, such as dominance and epistatic effects, play an important role in semen production and should therefore be considered in genetic improvement programs. In this study, the repeatability of semen production traits in Japanese Black bulls (JB) as beef bulls and Holstein bulls (HOL) as dairy bulls was evaluated by considering additive and nonadditive genetic effects using the Illumina BovineSNP50 BeadChip. We also evaluated the advantage of using more complete models that include nonadditive genetic effects by comparing the rank of genotyped animals and the phenotype prediction ability of each model. In total, 65,463 records for 615 genotyped JB and 48,653 records for 845 genotyped HOL were used to estimate additive and nonadditive (dominance and epistatic) variance components for semen volume (VOL), sperm concentration (CON), sperm motility (MOT), MOT after freeze-thawing (aMOT), and sperm number (NUM). In the model including both additive and nonadditive genetic effects, the broad-sense heritability (0.17 to 0.43) was more than twice as high as the narrow-sense heritability (0.04 to 0.11) for all traits and breeds, and the differences between the broad-sense heritability and repeatability were very small for VOL, NUM, and CON in both breeds. A large proportion of permanent environmental variance was explained by epistatic variance. The epistatic variance as a proportion of total phenotypic variance was 0.07 to 0.33 for all traits and breeds. In addition, heterozygosity showed significant positive relationships with NUM, MOT, and aMOT in JB and NUM in HOL, when the heterozygosity rate was included as a covariate. In a comparison of models, the inclusion of nonadditive genetic effects resulted in a re-ranking of the top genotyped bulls for the additive effects. Adjusting for nonadditive genetic effects could be expected to produce a more accurate breeding value, even if the models have similar fitting. However, including nonadditive genetic effects did not improve the ability of any model to predict phenotypic values for any trait or breed compared with the predictive ability of a model that includes only additive effects. Consequently, although nonadditive genetic effects, especially epistatic effects, play an important role in semen production traits, they do not improve prediction accuracy in beef and dairy bulls.

Improving reproductive efficiency is a key objective in the beef and dairy cattle industries, and bull fertility is an important determinant of the reproductive performance of cows. The heritability of semen production traits is generally low; however, their repeatability is moderate. This difference between repeatability and heritability suggests that nonadditive genetic effects, such as dominance and epistatic genetic effects, could have an important role in semen production traits in bulls. Here, we estimated repeatability for semen production traits in beef and dairy bulls by considering additive and nonadditive genetic effects. Our results suggest that the contribution of nonadditive genetic effects to differences between repeatability and heritability was very high. Nonadditive genetic effects, especially epistatic effects, played important roles in semen production traits in beef and dairy bulls. However, we found that the inclusion of nonadditive genetic effects in a predictive model does not improve phenotypic prediction accuracy; further studies are needed to improve the predictive ability when using nonadditive genetic effects.

L-amino acid oxidase 1 in sperm is associated with reproductive performance in male mice and bulls.

Sperm quality is an important indicator of male fertility, and a suitable biomarker enables the selection of high-quality spermatozoa. We previously found that L-amino acid oxidase encoded by the L-amino acid oxidase 1 (Lao1) gene exerts biological roles in the mammary gland and brain by converting specific L-amino acids into keto acids, ammonia, and hydrogen peroxide (H2O2). Here, we describe the role of Lao1 in male reproduction. Lao1-deficient (Lao1-/-) male mice generated fewer pregnant embryos and pups as well as lower ratios of fertilized oocytes and even ovulated number was not different, suggesting that male subfertility caused the smaller litters. We found that LAO1 expressed in acrosomes is associated with high malformation ratios and low viability of Lao1-/- sperm. Wild-type (WT) sperm produced more H2O2 than Lao1-/- sperm, and 10 µM H2O2 restored knockout (KO) sperm viability in vitro. In addition, the sperm ratio of induced acrosome reaction was higher in WT than in Lao1-/- sperm incubated with the calcium ionophore A23187. Moreover, LAO1 expression was abundant in bovine sperm with high fertilization ratios. We concluded that LAO1 localized in the sperm acrosome influences sperm viability and morphology as well as the acrosome reaction, and that LAO1-deficient sperm might cause male subfertility. Thus, LAO1 might serve as a novel marker for selecting high-quality spermatozoa, especially for livestock reproduction.

Functional insulin-like factor 3 (INSL3) hormone-receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility.

Insulin-like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone-receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he-goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he-goats. In comparison to fertile bulls, the percentage of INSL3- and RXFP2-expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3-binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone-receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.

Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality.

Sperm DNA damage affects the conception rate resulting from human assisted reproduction technology. The objective of this study was to adapt the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to provide a quality parameter for bull semen based on the detection of sperm DNA damage. Fresh semen was collected from two Japanese Black bulls (A, B) several times over the course of a year, and the percentage of TUNEL-positive spermatozoa (sperm TUNEL index) was determined. Individual differences in semen were detected using the sperm TUNEL index in these bulls (P < 0.01). The sperm TUNEL index of cryopreserved semen obtained from test-mated Japanese Black (n = 30, including two bulls with a conception rate lower than 10%) and Holstein (n = 34) bulls were analyzed. The average sperm TUNEL index and conception rate resulting from artificial insemination (AI) were 4.7% and 55.7% for Japanese Black, and 4.9% and 39.5% for Holstein, respectively. A weak negative correlation between sperm TUNEL index and conception rate was observed in Holstein bulls (P < 0.05). Semen samples from six bulls with more than 10% sperm TUNEL index were studied, and these samples showed low sperm viability. However, semen resulting in a very low conception rate did not have a high sperm TUNEL index. Although it would be difficult to predict a low conception rate resulting from AI using the sperm TUNEL index alone, the index can be used as an additional parameter to provide a more comprehensive description of semen quality.

Recombinant sea urchin flagellar adenylate kinase.

Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.

The 10 sea urchin receptor for egg jelly proteins (SpREJ) are members of the polycystic kidney disease-1 (PKD1) family.

BACKGROUND: Mutations in the human polycystic kidney disease-1 (hPKD1) gene result in ~85% of cases of autosomal dominant polycystic kidney disease, the most frequent human monogenic disease. PKD1 proteins are large multidomain proteins involved in a variety of signal transduction mechanisms. Obtaining more information about members of the PKD1 family will help to clarify their functions. Humans have five hPKD1 proteins, whereas sea urchins have 10. The PKD1 proteins of the sea urchin, Strongylocentrotus purpuratus, are referred to as the Receptor for Egg Jelly, or SpREJ proteins. The SpREJ proteins form a subfamily within the PKD1 family. They frequently contain C-type lectin domains, PKD repeats, a REJ domain, a GPS domain, a PLAT/LH2 domain, 1-11 transmembrane segments and a C-terminal coiled-coil domain. RESULTS: The 10 full-length SpREJ cDNA sequences were determined. The secondary structures of their deduced proteins were predicted and compared to the five human hPKD1 proteins. The genomic structures of the 10 SpREJs show low similarity to each other. All 10 SpREJs are transcribed in either embryos or adult tissues. SpREJs show distinct patterns of expression during embryogenesis. Adult tissues show tissue-specific patterns of SpREJ expression. CONCLUSION: Possession of a REJ domain of about 600 residues defines this family. Except for SpREJ1 and 3, that are thought to be associated with the sperm acrosome reaction, the functions of the other SpREJ proteins remain unknown. The sea urchin genome is one-fourth the size of the human genome, but sea urchins have 10 SpREJ proteins, whereas humans have five. Determination of the tissue specific function of each of these proteins will be of interest to those studying echinoderm development. Sea urchins are basal deuterostomes, the line of evolution leading to the vertebrates. The study of individual PKD1 proteins will increase our knowledge of the importance of this gene family.

Adenylate kinase in sea urchin embryonic cilia.

Sea urchin embryos swim by ciliary movement. Hypertonic shock causes deciliation and loss of motility. Within 2-4 h, cilia regenerate and the embryos swim again. Regeneration of cilia occurs multiple times. The adenylate kinase (AK) activity of isolated cilia was studied. A 130-kDa Sp-AK isozyme, present in sperm flagella, is also present in embryonic cilia. AK activity is responsible for approximately 93% of nonmitochondrial ATP regeneration from ADP in embryonic cilia. This is unlike sea urchin sperm flagella, where approximately 31% of the nonmitochondrial ATP regeneration is from the 130-kDa Sp-AK isozyme and approximately 69% from the flagellar creatine kinase (Sp-CK). Embryos were deciliated 1-3 times and after a 2-h period of regeneration the major ciliary axonemal proteins such as the tubulins appeared constant in amount. However, a moderate decrease in ATPase activity, and a large decrease of total AK activity, were measured. The decrease in AK activity paralleled the decrease in embryo swimming velocity. Embryos were deciliated once and cilia regeneration followed for 4 h. ATPase activity recovered to control levels by 3 h, but AK activity and swimming velocity remained lower than in controls. Detergent solubility data and kinetic experiments indicate that, in addition to the 130-kDa Sp-AK, there is at least one additional AK isozyme in embryonic cilia. Analysis of the S. purpuratus genome indicates five AK isozymes in addition to the 130-kDa Sp-AK isozyme. Decreased swimming velocity of embryos with regenerated cilia suggests that regenerated cilia are not as functionally perfect as naturally grown cilia.

A sea urchin sperm flagellar adenylate kinase with triplicated catalytic domains.

The mitochondrion of sea urchin sperm is located at the base of the sperm head, and the flagellum extends from the mitochondrion for approximately 40 microM. These sperm have two known flagellar, non-mitochondrial, enzymatic systems to rephosphorylate ADP. The first involves the phosphocreatine shuttle, where flagellar creatine kinase (Sp-CK) uses phosphocreatine to rephosphorylate ADP. The second system, studied in this report, is adenylate kinase (Sp-AK), which uses 2 ADP to make ATP + AMP. Cloning of Sp-AK shows that, like Sp-CK, Sp-AK has three catalytic domains. Sp-AK localizes along the entire flagellum, and most of it is tightly bound to the axoneme. Sp-AK activity and flagellar motility were studied using demembranated sperm. The specific Sp-AK inhibitor Ap5A blocks enzyme activity with an IC50 of 0.41 microM. In 1 mm ADP, flagella reactivate motility in 5 min; 1 microM Ap5A completely inhibits this reactivation. No inhibition of motility occurs in Ap5A when 1 mm ATP is added to the reactivation buffer. The pH optimum for Sp-AK is 7.7, an internal pH at which sperm are fully motile. The pH optimum for Sp-CK is 6.7, an internal pH at which sperm are immotile. In isolated, detergent-permeabilized flagella, assayed at pH 7.6, the Km for Sp-AK is 0.32 mm and the Vmax is 2.80 microM ATP formed/min/mg of protein. When assayed at pH 7.6, the Sp-CK Km is 0.25 mm and the Vmax 5.25. At the measured in vivo concentrations of ADP of 114 microM, at pH 7.6, the axonemal Sp-AK could contribute approximately 31%, and Sp-CK 69%, of the total non-mitochondrial ATP synthesis associated with the demembranated axoneme. Thus, Sp-AK could contribute substantially to ATP synthesis utilized for motility. Alternatively, Sp-AK could function in the removal of ADP, which is a potent inhibitor of dynein ATPase.

Roles of cAMP in regulating microtubule sliding and flagellar bending in demembranated hamster spermatozoa.

To understand the mechanism regulating spermatozoa motility, it is important to investigate the mechanism regulating the conversion of microtubule sliding into flagellar bending. Therefore, we analyzed microtubule sliding and its conversion into flagellar bending using a demembranated spermatozoa model in which microtubule sliding and flagellar bending could be analyzed separately by treating the demembranated spermatozoa with and without dithiothreitol, respectively. Using this model, we examined the roles of cAMP and its target molecules in regulating flagellar bending and microtubule sliding. Although flagellar bending did not occur in the absence of cAMP, microtubule extrusion occurred without it, suggesting that cAMP is necessary for the conversion of microtubule sliding into flagellar bending, but not for microtubule sliding itself. The target of cAMP for regulating flagellar bending was not cAMP-dependent protein kinase (PKA), since flagellar bending was still observed in the spermatozoa treated with a PKA-specific inhibitor. Alternatively, the Epac/Rap pathway may be the target. Epac2 and Rap2 were detected in hamster spermatozoa using immunoblotting. Since Rap2 is a GTPase, we investigated the flagellar bending of demembranated spermatozoa treated with GTPgammaS. The treatment markedly increased the beat frequency and bending rate. These results suggest that cAMP activates the Epac/Rap pathway to regulate the conversion of microtubule sliding into flagellar bending.

Analysis of flagellar bending in hamster spermatozoa: characterization of an effective stroke.

The mechanism by which flagella generate the propulsive force for movement of hamster spermatozoa was analyzed quantitatively. Tracing points positioned 30, 60, 90, and 120 microm from the head-midpiece junction on the flagellum revealed that they all had zigzag trajectories. These points departed from and returned to the line that crossed the direction of progression. They moved along the concave side (but not the convex side) of the flagellar envelope that was drawn by tracing the trajectory of the entire flagellum. To clarify this asymmetry, the bending rate was analyzed by measuring the curvatures of points 30, 60, 90, and 120 microm from the head-midpiece junction. The bending rate was not constant through the cycle of flagellar bending. The rate was higher when bending was in the direction described by the curve of the hook-shaped head (defined as a principal bend [P-bend]) to the opposite side (R-bend). We measured a lower bending rate in the principal direction (R-bend to P-bend). To identify the point at which the propulsive force is generated efficiently within the cycle of flagellar bending, we calculated the propulsive force generated at each point on the flagellum. The value of the propulsive force was positive whenever the flagellum bent from an R-bend to a P-bend (when the bending rate was lowest). By contrast, the propulsive force value was zero or negative when the flagellum bent in the other direction (when the bending rate was higher). These results indicate that flagellar bending in hamster spermatozoa produces alternate effective and ineffective strokes during propulsion.

Reducing agents induce microtubule extrusion in demembranated mammalian spermatozoa.

To understand the mechanism regulating flagellar bending in spermatozoa, it is important to investigate the regulation of microtubule sliding in the flagellar axoneme. It has been shown that protease treatment following demembranation with Triton X-100 disrupts the connections between microtubules and induces extrusion of microtubules from the flagellar axoneme. This approach enables a direct investigation of the regulation of microtubule sliding; however, the percentage of spermatozoa with protease-induced extrusion was relatively low, probably due to protease digestion of some regulatory motility proteins, as well as proteins connecting the microtubules. In this study, we demonstrate microtubule extrusion in most hamster and mouse demembranated spermatozoa upon treatment with a high concentration of the reducing agents dithiothreitol or 2-mercaptoethanol, without the use of proteases. The extrusion of microtubules occurred when the spermatozoa were treated with concentrations of the reducing agents that were sufficient for the reduction of the disulfide bonds of IgG. These results suggest that the arrangement of the axonemal structures connecting doublet microtubules depends to an important degree on -S-S- bonds. Close observation of the extrusion process using the present method revealed that microtubules were extruded on the same side as that of the curve of the sperm head, and also on the opposite side. Furthermore, we noted that extrusion always started on one side, followed by the other side, but was never initiated on both sides simultaneously.