Use of molecular marker diversity to increase forage yield in timothy (Phleum pratense L.)

Molecular marker technologies may provide a tool to overcome the forage yield plateau in timothy (Phleum pratense L.). Therefore, a study was designed to investigate the relationship between marker‐based genetic distance (GD) estimates and yield. Genetic distances were estimated using Dice coefficients framed by 28 simple sequence repeat markers. In Experiment 1, parents with high general combining ability (GCA) from two contrasting first‐generation synthetic strains (exhibiting high and low yields) were compared. Average GDs of each strain were 0.74 (high) and 0.68 (low). These differences indicated that GD may be partially the basis for contrasting yields. In Experiment 2, GDs among 40 parents of a polycross were used for calculating general genetic distance (GGD), which is indicative of their allelic complementation. Analyses revealed a significant (P < 0.01) moderate correlation (r) between GGD and GCA for yield (r = 0.45) and a significant (P < 0.01) residual mean square for the regression of yield on GGD, suggesting that considerable non‐additive effects were associated with GCA. The results are indicative of the potential use of GD estimation for yield improvement in timothy.     

Kinematic gait analysis and lactation performance in dairy cows fed a diet supplemented with zinc,manganese, copper and cobalt

This study investigated how supplementation of the diet of dairy cows with trace minerals (zinc, manganese, copper and cobalt) affected kinematic gait parameters and lactation performance. Eight Holstein cows were divided into two groups, with each group receiving a different dietary treatment (control diet, or control diet supplemented with trace minerals) in a two‐period crossover design. Kinematic gait parameters were calculated by using image analysis software. Compared to cows fed the control diet, cows that received the trace mineral‐supplemented diet exhibited significantly increased walking and stepping rates, and had a shorter stance duration. Feed intake and milk production increased in cows fed the trace mineral‐supplemented diet compared with control groups. The plasma manganese concentration was not different in control and experimental cows. In contrast, cobalt was only detected in the plasma of cows fed the supplemented diet. These results provide the first evidence that trace mineral supplementation of the diet of dairy cows affects locomotion, and that the associated gait changes can be detected by using kinematic gait analysis. Moreover, trace mineral supplementation improved milk production and only minimally altered blood and physiological parameters in dairy cows.     

Oxygen consumption rate of early pre-antral follicles from vitrified human ovarian cortical tissue

The study of human ovarian tissue transplantation and cryopreservation has advanced significantly. Autotransplantation of human pre-antral follicles isolated from cryopreserved cortical tissue is a promising option for the preservation of fertility in young cancer patients. The purpose of the present study was to reveal the effect of vitrification after low-temperature transportation of human pre-antral follicles by using the oxygen consumption rate (OCR). Cortical tissues from 9 ovaries of female-to-male transsexuals were vitrified after transportation (6 or 18 h). The follicles were enzymatically isolated from nonvitrified tissue (group I, 18 h of transportation), vitrified-warmed tissue (group II, 6 and 18 h of transportation) and vitrified-warmed tissue that had been incubated for 24 h (group III, 6 and 18 h of transportation). OCR measurement and the LIVE/DEAD viability assay were performed. Despite the ischemic condition, the isolated pre-antral follicles in group I consumed oxygen, and the mean OCRs increased with developmental stage. Neither the transportation time nor patient age seemed to affect the OCR in this group. Meanwhile, the mean OCR was significantly lower (P < 0.05) in group II but was comparable to that of group I after 24 h of incubation. The integrity of vitrified-warmed primordial and primary follicles was clearly corroborated by the LIVE/DEAD viability assay. These results demonstrate that the OCR can be used to directly estimate the effect of vitrification on the viability of primordial and primary follicles and to select the viable primordial and primary follicles from vitrified-warmed follicles.     

Production of bioactive bovine fibroblast growth factor 4 in E. coli based on the common nucleotide sequence of its structural gene in three breeds

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro³²‐Leu²⁰⁶) compared with the sequence previously reported. In the present study, HisbFGF4, a 6× histidine‐tagged bovine FGF4 (Pro³²‐Leu²⁰⁶), was produced in Escherichia coli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly²⁵‐Leu²⁰⁶) produced in E. coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E. coli utilizing its structural gene elucidated by us.     

Immunohistochemical localization of 3beta-hydroxysteroid dehydrogenase in the granulosa and theca interna layers of bovine cystic follicles

The aim of the present study was to determine whether the alteration of population of cells containing 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is responsible for the formation of cystic follicles. Paraffin sections of healthy (2 to 5 mm in diameter), atretic (2 to 5 mm) and cystic follicles (more than 25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine 3beta-HSD. The 3beta-HSD-positive cells were counted in 4 different regions of the follicles from the apical to the basal side. The frequencies of 3beta-HSD-positive granulosa cells in cystic follicles were significantly higher than those in the healthy follicles (P<0.05), although the number of 3beta-HSD-positive granulosa cells in the cystic follicle were fewer than half the cells (30 to 40%) and was much smaller than that in preovulatory follicles (Conley et al., 1995). The frequencies of 3beta-HSD-positive cells were higher in the granulosa layer and lower in the theca interna layer of the cystic follicles than the atretic follicles. These results suggest that the differentiation of granulosa cells to express 3beta-HSD might be insufficient in cystic follicles and accordingly they fail to ovulate. The differences of frequencies of 3beta-HSD-positive cells in the granulosa and theca interna layers between cystic and atretic follicles may be one of the reasons why regression is delayed in cystic follicles.     

Nutritionally induced body weight loss and ovarian quiescence in Shiba goats

Four female Shiba goats were used to determine the influence of body weight loss by dietary restriction on estrous cyclicity. The dietary restriction was started on the day following ovulation. The goats were fed hay cube and straw at an amount of 30% of energy requirement based on weekly body weight measurement. The ovaries were monitored daily by transrectal ultrasonography and blood samples were collected daily by jugular venipuncture for ovarian steroids analysis. After the start of food restriction, all animals lost body weight and entered ovarian quiescence. Intervals to the onset of ovarian quiescence tended to depend on the body weight of each animal at the start of food restriction. The mean concentration of progesterone during the mid-luteal phase (from 7 to 13 days after ovulation) in the last estrous cycle before ovarian quiescence was significantly lower than that in normal estrous cycle of the control period (19.7 +/- 2.8 vs 12.3 +/- 2.2 ng/ml, P<0.05), whereas there was no significant difference in the length of the luteal phase, determined as the period when corpora lutea existed and concentrations of progesterone were equal to or greater than 1 ng/ml (15.8 +/- 1.5 vs 15.0 +/- 2.8 days, P>0.1). A rise of estradiol concentration and follicular growth in the follicular phase following a decline of progesterone level after luteal regression tended to be suppressed at the onset of ovarian quiescence. It seems that the present results are consistent with previous findings that nutritionally induced body weight loss influences the secretion of ovarian steroids and eventually induces ovarian quiescence.     

Unilateral intrauterine insemination with cryopreserved caudal epididymal sperm recovered from refrigerated canine epididymides

Canine epididymides were excised and immediately stored at 4 degrees C for 48 hr, and the qualities of caudal epididymal sperm after recovery and cryopreservation were evaluated. To confirm the fertility of the cryopreserved caudal epididymal sperm, artificial intrauterine insemination was performed. The sperm motility (61.0%) immediately after recovery from caudal epididymis stored at 4 degrees C for 48 hr was significantly lower than those of sperm stored for 0 and 24 hr (88.6 and 80.7%, respectively), but there was no significant difference after freeze-thawing (0-, 24-, and 48-hr storage groups: 27.9, 24.3, and 28.3%, respectively). The incidence of abnormal sperm immediately after recovery was significantly higher in the 24-hr and 48-hr storage groups (19.3 and 27.7%, respectively) than in the 0-hr storage group (5.6%), and a significant difference was also observed after freeze-thawing. The incidence of immature sperm with cytoplasmic droplets was significantly higher in the 48-hr storage group (18.4%) than in the 0-hr storage group (4.7%), but there was no difference after freeze-thawing. By unilateral intrauterine insemination (2x10(8) sperm), 4 of 5 bitches (80%) conceived. The above findings demonstrated that sperm motility was good even enough the incidence of abnormal sperm was high in canine epididymal sperm that were recovered from the epididymis stored at 4 degrees C for 48 hr and cryopreseved, and that artificial intrauterine insemination resulted in a high conception rate.     

Expression of gap junction protein connexin 43 during follicular atresia in the ovary of swamp buffaloes

The present study was performed to detect the presence of gap junction protein connexin 43 (Cx43) and describe the changes in its expression during ovarian follicular atresia in the swamp buffalo in comparison with cattle. Ovaries of Philippine swamp buffaloes (Bubalus bubalis; SB) and Holstein-Friesian cows (Bos taurus; HF) were collected from slaughterhouses, fixed in 10% formalin in PBS and embedded in paraffin. Sections of healthy follicles and at various follicular stages of atresia were immunostained with anti-Cx43 antibody. Cx43 appeared as punctate staining between granulosa cells (healthy to advanced atretic follicles), indicating assembled gap junctions, but was absent in the theca interna. In SB as well as in HF, granulosa cells showed a dense, moderate, and sparse immunoreactivity to Cx43 in healthy, early atretic, and advanced atretic follicles, respectively. Cumulus cells (in the advanced atretic follicle) surrounding oocytes and adjacent granulosa layers retain the Cx43 protein, although there was only a sparse expression of Cx43 observed in the granulosa layers distant from oocytes in the same follicles. The results indicate that gap junction protein Cx43 decreases in association with atresia and supports the concept that a loss of gap junctional communication plays a coordinating role in the process of atresia. Furthermore, the schema of Cx43 immunoreactivity in SB granulosa cells is similar to that of HF.