Assessment of pubertal development of boars derived from ultrasonographic determination of testicular diameter

At the onset of puberty, seminiferous tubules rapidly increase in diameter, thereby occupying a greater proportion of the testis, resulting in a rapid increase in testicular size. The objective of the current studies was to evaluate ultrasonography for assessing testicular diameter, as a basis for ranking boars relative to their extent of pubertal development. In the initial study, prior to castration at 4, 5, 6, or 7 mo of age, testicular length and diameter were assessed by ultrasonography in 160 anesthetized boars. After castration, testes were weighed. Mean diameter of seminiferous tubules and percentage of the testis occupied by tubules were determined by histological evaluations of all testes. Testicular volume was calculated from length and diameter and was correlated with testicular weight (P < 0.001; r ≧ 0.78) within each of the four age groups. At 4 and 5 mo of age, testicular diameter correlated positively (P < 0.001) with diameter of seminiferous tubules; this relationship was not significant at older ages. In two subsequent studies, testicular diameter determined ultrasonographically in conscious boars was highly correlated (r > 0.8) when assessed twice on the same day, or when diameter of the right was compared with diameter of the left testis. Similarly, testicular diameter obtained initially at 92 d of age correlated positively (P < 0.001) with the diameter observed at older ages, but the magnitude of the relationship decreased as time between evaluations increased. These findings supported ultrasonographic determination of testicular diameter during early pubertal development, as a means to rank boars of similar chronological age for extent of pubertal development.     

B-mode ultrasonographic examination of the accessory sex glands of boars

Thorough examinations of the reproductive system of boars are generally not performed on normal boars to be used for breeding; only boars with problems undergo a form of a breeding soundness examination. In order for veterinarians to identify pathological conditions, the normal architecture of the accessory sex glands needs to be described. The purpose of this study was to use B-mode ultrasonography to describe the accessory sex glands in the boar and to see if transrectal ultrasonography would be a viable option in which to obtain this data. Initially, cross-sectional saline bath examinations of accessory sex glands were performed on crossbred boar reproductive tracts (n = 4) using B-mode ultrasonography equipped with a 5 MHz dual frequency linear array transducer. In situ examinations were also performed on terminal line crossbred boars (n = 16) ranging in age from 10 to 23 months old using the same ultrasound methodology; four boars were under general anesthesia and the remaining 12 were standing in crates. Eight boars were abstinent for 2 days and the other eight had ejaculates collected 2 h prior to examination. The paired bulbourethral glands are best described as a long oval gland with a uniformly echogenic appearance with a large anechoic space in the center of the gland extending most of its length. The walls of the vesicular glands were found to be thin, with the parenchyma having multiple small echolucent areas that appeared to merge and form a central canal. The prostate gland was best identified as a pecan-sized gland with a uniform echogenic appearance. Visualization of the prostate gland was accomplished with more proficiency using the saline bath ultrasonography as compared to in situ examinations. All of the accessory sex glands could be examined using both methodologies of ultrasonographic examination with a 5 MHz frequency linear array transducer. It was determined that each accessory sex gland could be recognized, and differences between ejaculated and nonejaculated boars could be identified. The results of this study demonstrate that transrectal ultrasonography can be used as a diagnostic aid in assessing the accessory sex glands of boars.     

Significance of total sperm numbers and microscopic evaluation of bull semen frozen in pellets in relation to fertility

During 1978 semen of 12 bulls was frozen in pellets containing 10 million and 30 million total sperm cells respectively. N.R. (non return) rates of the pellets containing 30 million sperm cells were 3.44% higher (P<0.001). Differences among bulls however vary considerably.No significant differences could be proven when pellets made of semen qualified as “very good” were used in both concentrations. The qualification “very good” was made after an incubation test when the ejaculates showed at least 70% estimated motile spermatozoa.     

Endocrine responses in relation to compensatory testicular growth after neonatal hemicastration in boars

Mass (TM) and relative mass (organ mass/body mass; RTM) of the right testis and epididymis (EM and REM, respectively) were determined every 14 days from 10 to 122 days of age for intact boars (I) and boars hemicastrated on Day 10 (HC) in two crossbred herds (Trial 1 and Trial 2). Plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH), and testosterone were determined in four blood samples from each pig, three collected 24 h prior to castration and one immediately prior to castration. Values for TM and RTM of HC boars were approximately double (p less than 0.0001) those of I boars by 38 days of age, and these differences were maintained through Day 122. Both EM and REM were greater (p less than 0.05) in HC than in I boars from Day 52 to Day 122. The TM, RTM, EM and REM were greater (p less than 0.05) in Trial 1 than in Trial 2 for both I and HC boars from Day 80 to Day 122, indicating an earlier onset of pubertal testicular growth in the Trial-1 boars. Plasma GH concentration was greater (p less than 0.05) in HC than in I boars from Day 16 to Day 38. A transient increase in plasma FSH (p less than 0.05) was observed from Day 24 to Day 38. After Day 38, there was no difference (p greater than 0.05) in FSH or GH between HC and I boars, or between trials. Plasma LH, prolactin, and testosterone concentrations were also similar in HC and I boars.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.     

Gonadal sperm reserve in purebred Landrace and Large White boars of high average daily gain

The present study investigated the effects of average growth rate (AGR) levels and age on the number of sperm cells per gram of testis parenchyma and on the gonadal reserve in Landrace (LD) and Large White (LW) boars. In Experiment 1, the effects of breed (LD, LW), level of AGR from birth up to 90 days of age (Level 1: 384 +/- 32 g/day; Level 2: 512 +/- 22 g/day; Level 3: 624 +/- 41 g/day), and age (13, 15, 17, 19 and 21 weeks) on testicular cell concentration were evaluated. Data were analyzed under a 2 x 3 x 4 factorial design. There were significant effects associated with breed (P < 0.001) and age (P < 0.001) but not with AGR (P > 0.05) on sperm cell number per gram of testicular parenchyma. The number of cells increased with age and was greater in LW than in LD young boars, mainly those up to 19 weeks of age. In Experiment 2, the effect of two AGR levels (Level 1: 649-694 g/day; Level 2: 813-885 g/day) from birth up to 100 kg body weight on the number of sperm cells per gram of testis parenchyma and on the gonadal reserve was investigated using 59 purebred LD and LW boars. The boars were castrated at 23, 25, 29 and 33 weeks of age. Age of boars significantly affected gonadal sperm reserve and the number of sperm cells per gram of testicular tissue (P < 0.001). Breed of boars and AGR Levels did not significantly affect number of sperm cells and gonadal sperm reserve (P > 0.05). It was concluded that the number of sperm cells in the testicular tissue of young boars is influenced by their breed and age, but not by the level of their AGR.     

Flow cytometric evaluation of sperm parameters in relation to fertility potential

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.     

Pachytene pairing in relation to sperm and oocyte numbers in a male-fertile reciprocal translocation in the mouse

In adult males carrying the male-fertile reciprocal translocation T(2;4)13H, body weights, testis weights, and sperm counts were higher in heterozygotes than in homozygotes. Heterozygotes whose mothers were C3H/He exceeded their reciprocal counterparts in the same criteria. At 3-4 days of age, no significant differences between homozygous and heterozygous females were found in body weight, ovarian volume, or oocyte numbers, although mean oocyte volumes were somewhat larger in heterozygotes than in homozygotes. In homozygous males and females the synaptonemal complexes of rearranged chromosomes appeared as bivalents that were indistinguishable from normal bivalents. In most gametocytes of heterozygotes, the translocation was present in the form of a quadrivalent. The degree of pairing failure was greater in oocytes than in spermatocytes. Terminal asynapsis of quadrivalents was very rare in spermatocytes, but it affected one quarter of the oocytes. Only very few translocation configurations were associated with the XY bivalent. It is concluded that the number of sperm produced in male heterozygotes can match the general increase in vigor by the formation of a high level of fully paired quadrivalents, whereas a greater degree of terminal asynapsis in the quadrivalents of oocytes may indicate a slightly more deleterious effect of this translocation on oogenesis.     

Validation of the sperm mobility assay in boars and stallions

The sperm mobility assay used in the present study measures the rate of sperm penetration in a biologically inert cell-separation solution (Accudenz). When a sample of sperm is overlaid in a cuvette containing Accudenz, sperm penetrate the solution and absorbance of the sample can be measured with a spectrophotometer. This assay has been successfully used to select chicken and turkey semen donors. We validated this assay for semen from boars and stallions. Absorbance was measured after overlaying fresh semen from each species in prefilled cuvettes for 1, 5, 10, 15, 20, and 40 min. There were no significant differences when sperm were incubated in prewarmed cuvettes at 37, 39, or 41 degrees C. However, a minimum concentration of 5x10(7) viable sperm/mL was needed to evaluate the rate of sperm penetration in boars. Absorbance was half-maximal at 5.4 and 14.1 min for boar and stallion sperm, respectively. Frequency analysis suggested a normal distribution of mobility values for boar sperm. There were positive correlations between mobility values and several computer-aided sperm analysis (CASA) parameters. In addition, there was medium repeatability for multiple ejaculates from single males. We concluded that the mobility assay can be used for mammalian sperm and there seemed to be phenotypic variation among boars in mobility estimates.     

Reproductive development in young boars exposed to sexually mature, nonpregnant sows and gilts

Thirty Yorkshire boars were allotted by litter to one of two treatment groups. Fifteen boars were reared, from 14 to 22 wk of age, in fenceline contact with mature sows and gilts that were randomly exhibiting estrus. Fifteen littermates of these boars, reared without exposure to females, served as controls. Boar-to-boar sexual behaviors were observed 1 hour daily from 16 to 22 wk of age. At ages 14 to 30 wk, boars were weighed at 4-wk intervals. Five boars per treatment were slaughtered when 22 wk old and twelve others (six per treatment) when 30 wk old. Vesicular gland, bulb of the prostate gland, bulbourethral gland, and each testis, cauda epididymis, and combined caput and corpus epididymis were closely trimmed and weighed. Left cauda epididymal sperm and left testis elongated spermatids were determined by homogenization and hemacytometer counting. At 26, 30, and 38 wk of age, boars were tested for mating efficiency by exposure to an estrous gilt. Boars exposed to females tended to be lighter at 22 wk and were lighter at 30 wk (P<.05). Total male-to-male sexual acts from 16 to 22 wk were correlated with mating-efficiency score at 26 and 30 wk (r = .69 and .68, respectively; (P<.05)). Correlation between testicle size and total testicle elongated spermatids was high at 30 wk of age (r = .92; P<.001). Results indicated that exposing young boars to mature females had limited effects on the boars' reproductive development; however, there were strong indications of a relationship between boar behavior during rearing and postpuberal mating performance.     

Stem diameter in relation to plant water status

An instrument containing a linear variable differential transformer was constructed to obtain continuous, nondestructive measurements of both short term changes in stem diameter and long term growth. In cotton plants, stem diameter, leaf water potential, and leaf relative water content are all closely related to net radiation at the top of the canopy. Leaves from the east and west sides of a plant show slight, but consistent differences in diurnal water potential patterns.     

Bovine oocyte diameter in relation to developmental competence

This study was conducted to determine the diameter of bovine oocytes that were able to attain their full developmental competence to blastocysts. Oocytes were recovered by aspiration of surface-visible follicles (1 to 7 mm in diameter) from slaughterhouse ovaries. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and they were divided into six groups based on diameter: < 110 microm, 110 to < 115 microm, 115 to < 120 microm, 120 to < 125 microm, 125 to < 130 microm and >/= 130 microm. Oocytes were processed through standard procedures for in vitro maturation, fertilization and culture. Following in vitro maturation or fertilization, some oocytes were stained to assess nuclear maturation and penetration rates. The numbers of embryos that cleaved at 42 h post insemination and developed to blastocysts and hatched blastocysts after 8 days of culture were recorded. The mean oocyte diameters were 114.0 +/- 4.8 microm. The oocytes displayed size-related ability to undergo meiotic maturation. The rates of nuclear maturation of oocytes in the greater than 115-microm size range were significantly higher than those of oocytes with diameters < 115 microm. In the < 120 microm diameter groups, the polyspermic fertilization rates of oocytes < 115 microm were significantly higher than those of oocytes 115 to < 120 microm in diameter. The rates of cleavage and development to blastocysts and hatched blastocysts rose as oocyte diameter increased. Among oocytes with a diameter >110 microm, oocytes < 120 microm were found to have significantly lower developmental competence than oocytes 120 to < 130 microm in diameter. These results suggest that bovine oocytes have acquired full meiotic competence at a diameter of 115 microm but not yet attained full developmental competence to blastocysts, and that oocytes have acquired full developmental competence at a diameter of 120 microm.     

Early sexual maturity in local boars of Northeastern India: age-related changes in testicular growth, epididymal sperm characteristics and peripheral testosterone levels

The present study reports the age related changes in the peripheral testosterone levels, testicular and epididymal growth and development and cauda epididymal spermiogram in local pigs of Northeastern India, which attain sexual maturity around 3 months of age. Local boars (n = 20) were castrated at monthly intervals from 2 to 6 months of age (4 boars per month) to study the testicular growth and development and the epididymal spermiogram. Blood samples, collected from local boars (n = 6) at monthly intervals from 2 to 6 months of age, were analyzed for testosterone levels by radioimmunoassay. Compared to Hampshire boars, significantly (P < 0.05) high testosterone levels were observed in the local boars as early as 2 months of age. The mean (± SEM) level of testosterone in the local boars at 2, 3 and 4 months of age was 11.89 ± 1.52, 20.45 ± 1.33 and 20.38 ± 2.0 ngml⁻¹, respectively. Though there was consistently significant (P < 0.05) difference in the body weight between Hampshire and local pigs, the same was not observed in case of testicular weight except at 3 and 6 months of age. In line with the above observation, the testis:body weight ratio (gram testis per kg body weight) was significantly (P < 0.05) higher in the local boars compared to the Hampshire boars at any time of observation, which ranged from 0.8 to 1.0 in case of Hampshire and from 2.3 to 3.0 in local boars. The sperm concentration in the cauda epididymal fluid of local boars at 2, 3 and 6 months of age was 2255 ± 186.6, 3685 ± 103.8 and 4325 ± 146.2 million/ml, respectively and the sperm motility, viability and total abnormality was 73.3, 75.2 and 6.2%, respectively at 3 months of age. Taken together, the testosterone level, testicular growth and development and epididymal spermiogram indicate the trait of early sexual maturity in the local pigs as compared to Hampshire.     

Morphofunctional alterations in testicular cells of deslorelin-treated boars: an immunohistochemical study

In this study we thoroughly scrutinized testes morphology and investigated whether treatment of recipient boars with gonadotropin-releasing hormone (GnRH)-agonist deslorelin could alter the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), luteinizing hormone receptors (LHRs), and androgen receptors (ARs) in testicular cells. An implant containing 4.7 mg of the GnRH-agonist deslorelin was subcutaneously inserted into crossbred male pigs at 91 and 147 days of age. Testicular traits, morphology of the testes, the proteins' expression, and testosterone concentration in blood plasma were analyzed in all boars after slaughter at 175 days of age. Histological analysis revealed significant alterations in both the interstitial tissue and seminiferous tubules of experimental animals after 28 and 84 days of deslorelin treatment. The intensity of the AR immunostaining within the testis appeared as a function of the severity of testicular dysgenesis. Time-dependent action of deslorelin on the expression of LHR and 3beta-HSD in Leydig cells was also detected. Staining for LHR and 3beta-HSD was very weak or the Leydig cells were immunonegative. Concomitantly, a significant decrease in plasma testosterone level was found in both groups of deslorelin-treated boars when compared with the control group. This is the first report showing the cellular distribution of AR, LHR, and 3beta-HSD in testicular cells of deslorelin-treated boars. It is concluded that morphological and immunohistochemical studies are important for the evaluation of testicular histoarchitecture and steroidogenic function. Subsequently, the endocrine control of reproduction in the GnRH-agonist deslorelin-treated males will be better understood.     

Quantification of spermatozoa in the sperm-storage tubules of turkey hens and the relation to sperm numbers in the perivitelline layer of eggs

This study was conducted to determine the number of spermatozoa residing in the oviduct sperm-storage tubules (SST) and the relationship between these numbers and the number of spermatozoa embedded in the perivitelline layer of oviductal eggs after a single insemination of 200 x 10(6) spermatozoa. The SST of hens inseminated within one week before the expected onset of egg production were filled faster (4 h vs. 2 days) and possessed more spermatozoa (4.1 vs. 2.0 x 10(6)) than the SST of hens inseminated after the onset of egg production. Furthermore, for hens in egg production, significantly fewer spermatozoa were recovered from the SST if the hen was inseminated within 2 h before or after oviposition than if inseminated more than 2 h before or after the oviposition. There was a strong positive correlation between the number of spermatozoa in the SST and the number of spermatozoa embedded in the perivitelline layer of the oviductal eggs (r = 0.85, p less than 0.01). These data show that the population of spermatozoa actually accepted by the SST is quite small relative to the number of spermatozoa inseminated and that maximum sperm-storage is achieved when the hen is inseminated just prior to the onset of egg production. It is suggested that the sperm-storage capacity of the oviduct and the quality of the semen sample can be estimated on the basis of numbers of spermatozoa embedded in the egg perivitelline layer.     

Incomplete translocations induced in mature sperm of Drosophila

Frequencies of incomplete translocations in subcentromeric heterochromatin induced by gamma-rays in mature sperm were estimated in the "two right acrocentrics and left compound" system of Drosophila chromosome 2. The value obtained at 3000R was (2.5-2.9) X 10(-3). Apart from incomplete translocations leading to formation of new metacentrics, acrocentrics were also formed. It is suggested that they appear via reunion of fragments after replication of paternal chromosomes. Incomplete translocation were shown to be formed as a result of loss of centric heterochromatin fragment of acrocentric and acentric arm of the compound, as opposed to formation, due to loss of acentric heterochromatin fragment of acrocentric and centric fragment of the compound. Analysis of the dose-effect curve for half-translocations show that the latter are two hit events.     

Estimation of sperm numbers in insects by fluorometry

Summary To facilitate the study of mating biology in the desert leaf-cutter antAcromyrmex versicolor, methods were developed that allowed storage and easy quantification of sperm samples collected from both male and female reproductive tracts. Sperm samples stored frozen were sonicated, stained with a fluorescent DNA stain, and the fluorescence emitted by the stained sperm heads was measured. The intensity of fluorescence was shown to be a linear function of the number of sperm in the sample as determined by counting.