Infertility in a bull with a nuclear sperm defect: A case report

A Simmental bull with a history of low fertility, both by natural service and artificial insemination, was presented for examination. Two previous semen evaluations had revealed no specific semen abnormalities that would support the breeding history. A comprehensive cytochemical analysis of the bull's ejaculate revealed a complex nuclear lesion affecting over 80% of sperm. This condition was expressed in abnormal shaping of the nuclei, with deficient distribution, condensation and stabilization of the nucleoplasm. These abnormalities were associated with various-sized intranuclear pouches or depressions. The acrosome was moderately involved and the tail was relatively free of abnormalities resulting in normal sperm motility.Two controlled breeding trials utilizing a total of 15 super-ovulated females were conducted to evaluate the bull's fertilization rate. Combined data demonstrated an 18% ($\text{23}\text{128}$) fertilization rate of recovered ova. At the same time, the fertilization rate of seven bulls classified as satisfactory potential breeders was 72% ($\text{353}\text{490}$).Data from two embryo transplant units regarding ova collected from eight donor females inseminated with semen from this bull revealed a fertilization rate of 41% ($\text{30}\text{73}$). Of the fertilized ova, 37% ($\text{11}\text{30}$) were degenerate and were not transferred. A pregnancy rate of 57% ($\text{11}\text{19}$) resulted from the transfer of 19 fertilized ova.A natural breeding pregnancy rate of 5% ($\text{2}\text{42}$) and artificial breeding pregnancy rate of 8% ($\text{15}\text{180}$) support the breeding trial results.     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.     

Diagnosis of trichomoniasis in pregnant cows by culture of cervical-vaginal mucus

The objective of this study was to evaluate the effectiveness of Cervical-Vaginal Mucus (CVM) cultures for isolating $\text{Trichomonas fetus}$ from pregnant cows. Cervical-vaginal mucus was cultured from sixty-eight cows averaging 98 days of gestation. $\text{Trichomonas fetus}$ was isolated from 7 of 36 cows in groups exposed to bulls found to be infected at the end of the breeding season. The remaining 32 cows which were bred to bulls found not to be infected at the end of the breeding season were negative. Seven of the cows serviced by infected bulls aborted. Three of these had been positive and 4 negative on culture. The incidence of abortion was determined by examination 81 days after the previous examination and CVM culture. During the same interval there were no abortions in the 32 cows served by non-infected bulls. Culture of CVM identified infected groups of cows which had a high risk for abortion.     

Trypanosoma lewisi: antibody classes which mediate precipitation, agglutination, and the inhibition of reproduction

Sera from Trypanosoma lewisi-infected and uninfected rats were applied to Protein A-Sepharose CL-4B columns. The absorbed fractions of antisera which contained only IgG molecules were reacted in microimmunodiffusion analyses with the exoantigens of T. lewisi in plasma collected from irradiated infected rats, and formed one precipitin line. These sera were also applied to T. lewisi extract immunoabsorbent columns and bound proteins were eluted and analyzed by immunodiffusion against antisera specific for rat immunoglobulins. IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM were absorbed by the immuno-absorbent columns. Absorption of the rat antisera with anti-rat IgG or anti-rat IgM removed one of the two precipitin lines against extracts prepared from parasites collected from irradiated infected animals. The absorbed IgG fractions and nonabsorbed fractions of antisera which were collected after Protein A-Sepharose CL-4B column chromatography agglutinated trypanosomes. After treatment of antisera with 2-mercaptoethanol, the agglutinin titers were lower than those of the control antisera suggesting both IgG and IgM are involved in the agglutination. The ablastic activity of the fractions eluted from Protein A-Sepharose CL-4B Chromatographic columns was assayed in cultures of bloodstream forms ofT. lewisi. Ablastic activity of proteins of antisera absorbed by the columns was demonstrated indicating they belonged to the IgG class of antibodies.     

Bovine trichomoniasis: Results of a slaughter survey in Colorado and Nebraska

A total of 2,909 bulls was cultured for the presence of Trichomonas fetus at slaughter at 4 abattoirs in Colorado and Nebraska. Samples were collected using the dry pipette preputial scraping technique and were transported to diagnostic laboratories within 24 h. Of the bulls cultured, 5 were positive. The state of the last origin for each animal was Kansas, 1 bull, Montana, 1 bull, Nebraska, 1 bull, and Utah, 2 bulls. These results indicate and incidence of less than 1% (0.172%) of trichomoniasis in the populations tested, even in the states of Colorado and Nebraska where the larger numbers of bulls were cultured.     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

Hymenolepis microstoma: Mouse strain differences in resistance to a challenge infection

Antibody responses and host resistance to the tapeworm, Hymenolepis microstoma, were investigated using $\text{AKR}\text{J}$ and $\text{C}\text{3}\text{HeB}\text{FeJ}$ strains of mice. AKR mice were significantly more resistant than controls to a secondary infection following exposure to a 3-, 21-, or 40-day primary infection. During a primary infection, intestinal anti-worm antibody responses measured by an enzyme-linked immunosorbent assay were elevated in the more resistant AKR strain, whereas serum antibody titers did not differ between the two strains. However, during a secondary infection, serum IgA titers were higher in AKR mice than C3H mice. Suppression of the serum IgA anti-worm response by oral administration of lipopolysaccharide also suppressed resistance to a secondary infection. Intraperitoneal immunization with worm antigen resulted in a minor degree of protection in AKR mice. This protection was associated with increased intestinal antibody titers compared to mice not demonstrating protection. These results suggest that the protective responses observed in AKR mice relative to C3H mice reflect differences in mucosal antibody responses to H. microstoma.     

Release of LH after administration of an analog of synthetic LH-RH in cattle

The $\text{in vivo}$ effect of an analog of LH-RH, (Des-Gly-NH₂¹⁰, Proethylamide⁹)-LH-RH, on LH release, was studied in one bull with azooapermia, and one cow with cystic follicle. The single intravenous injection of 100 μg LH-RH analog caused abrupt and marked increase in serum LH and the high levels of LH were maintained for 3 hours. The highest levels of serum LH were 48 and 114 times as high as the pre-treatment levels in a bull and a cow, respectively. Further studies are being undertaken to evaluate this compound for improving reproductive function in cattle.     

Reproductive studies of Brahman cattle I. Behavioral effect of various dose levels of Estradiol-17β upon ovariectomized Brahman,Brahman × Hereford and Hereford cows

Six ovariectomized mature cows each of Brahman (B), Brahman × Hereford (BH) or Hereford (H) breeding were injected intramuscularly with Estradiol-17β (E). Dose levels of 1, 2, 4 and 8 mg E were given in 2 ml corn oil. Cows were allowed a 2 week recovery period between treatments. After injection the cows were placed with 18 epididymectomized bulls and observed constantly for 36 hours. B failed to accept the bulls at any E dose level. Proportions of BH accepting the bull were $\text{2}\text{6}$, $\text{6}\text{6}$, $\text{6}\text{6}$ and $\text{6}\text{6}$ and proportions of H accepting the bull were $\text{5}\text{6}$, $\text{6}\text{6}$, $\text{5}\text{6}$ and $\text{6}\text{6}$ at 1, 2, 4 and 8 mg E, respectively. BH were less responsive at 1 mg E than H (P<.10) and B were less responsive at any level (P<.005). The number of stances increased significantly with dose level (P<.005) and a breed × dose level interaction (P<.10) was found. The duration of standing estrus behavior was longer in H cows at 1 and 2 mg E than in BH (P<.05) but was identical at 4 and 8 mg E. Duration of estrus was shorter in B except at the 2 mg dose level. Breed (P<.005) and breed × dose level interactions (P<.05) were found. Response time (injection of E to stance event) did not differ between dosages of E within breed groups. However, response time was significantly longer in B (19.3 hrs, P<.05) versus the response time of either H (10.1 hrs) or BH (12.8 hrs). If homosexual stance behavior is accepted as estrus, B were less responsive at 1, 2, 4 and 8 mg E than were BH or H (P<.10).     

The relationship between 13, 14-dihydro-15-keto PGF2α and LH secretion in bulls

Half-life ($\text{t}\text{1}\text{2}$), volume of distribution (Vd)_and total body clearance (TBC) of 13, 14-dihydro-15-keto PGF_2α (PGFM) were measured in order to determine optimal sampling frequency for accurate measurement of PGFM. Three yearling Holstein bulls (349.2 ± 6.7 kg) and 3 yearling Holstein steers (346.7 ± 7.0 kg) were utilized in a 3 × 3 Latin square design. Animals were given 0, 25 or 50 μg PGF_2α I.V.; blood samples collected every 2 min and plasma PGFM determined. The $\text{t}\text{1}\text{2}$, Vd and TBC of PGFM were 2.3 ± .2 min, 43.3 ± 3.3 liters and 13.7 ± 1.9 liters/min, respectively and were similar for 25 and 50 μg doses. To determine the relationship between endogenous PGFM and LH secretion in bulls, blood samples were collected every 2 min for 12 h in 4 yearling Angus bulls (489.1 ± 11.6 kg). All animals elicited at least one LH surge and PGFM concentrations were measured in samples coincident with the LH surge. Mean plasma PGFM concentrations were greater prior to the LH surge than during the LH surge. In addition, mean plasma PGFM concentration and frequency of PGFM peaks appeared to increase prior to the LH surge suggesting an association between PGFM and pulsatile LH secretion in the bull.     

On the defect of a dCMP hydroxymethylase mutant of bacteriophage T4 showing enzyme activity in extracts

Infection by $\text{L}\text{13}$, a temperature-sensitive mutant of gene 42 of phage T4, the structural gene for dCMP hydroxymethylase, previously was shown not to form T4 DNA at nonpermissive temperatures. Yet the enzyme activity was found in extracts. Since inactivation of the enzyme was not reversible, we have examined acid-soluble extracts of cells infected at nonpermissive temperature by $\text{tsL}\text{13}$ for 5-hydroxymethyldCMP in order to determine whether the enzyme functioned $\text{in vivo}$. A double mutant of $\text{tsL13}$ and $\text{amB}\text{24}$ (5-hydroxymethyldCMP kinase) did not form the nucleotide at nonpermissive temperature, but the control, $\text{amB}\text{24}$, formed large quantities. From these results and previous temperature-shift studies it is suggested that the enzyme is normally activated to function $\text{in vivo}$ between 5 and 8 minutes after infection.     

Indirect plaque-forming cells detected by use of normal mouse serum. I. Normal mouse serum plaque-forming cells are IgA producers.

The identity of indirect plaques developed upon addition of normal mouse serum (NMS) to Jerne test dishes was investigated. NMS plaques were significantly correlated with IgA, IgG_2A and IgG_2B plaques in decreasing order, but not with IgG₁ plaques. With protein A of Staphylococcus aureus as developer of IgG₂ plaques, the nonidentity of NMS plaque-forming cells (PFC) and IgG₂-producing cells could be demonstrated. The subsequent use in different orders of anti-α serum and NMS as developers of indirect plaques suggested that NMS PFC are IgA producers. Blocking experiments with purified myeloma IgA (MOPC 315) supplied further evidence for this idea. Using easily available protein A and NMS as developers of indirect plaques enables collection of more uniform data on the numbers of IgG₂ and IgA PFC than could be obtained with anti-heavy-chain sera.     

Separation of bovine spermatozoa proteins using 2D-PAGE revealed the relationship between tektin-4 expression patterns and spermatozoa motility

Poor semen quality has long been associated with bull infertility. However, the molecular basis in spermatozoa cells underlying the mechanisms of bull infertility remain unknown. The purpose of this study was to determine whether there is any protein in bovine spermatozoa related to semen quality. Semen samples from 18 Brahman bulls, 3 to 10 yrs of age, were assessed for semen quality in terms of spermatozoa motility and spermatozoa morphology. Spermatozoa extracts were separated using 2D-PAGE followed by staining with Coomassie blue. At least one duplicate gel was performed for each sample. Each gel was scanned with an ImageScanner System and analyzed for spots by ImageMaster 2D platinum software. The related protein spot(s) with semen quality was cut from the gel and identified by LC MS/MS. The results showed that at least 600 protein spots were detected in the spermatozoa extracts of the Brahman bulls. Of all these spots, there were 3 of 56 kDa at pI 6.4, 6.6 and 6.8 (Z₁, Z₂ and Z₃, respectively) that clearly showed different expression pattern among 18 Brahman bulls. Of 18 bulls (a) five showed the presence of spot Z₁ and Z₂ (pattern A) (b) one of spot Z₃ (pattern B) (c) five of spot Z₂ and Z₃ (pattern C) (d) one of spot Z₁ (pattern D) and (e) six of spot Z₂ (pattern E). Identification of spot Z₁, Z₂ and Z₃ by LC MS/MS had a similar result as matched to the tektin-4 protein of Bos taurus with a respective score of 171, 557 and 591. The statistical analysis of the 56 kDa protein patterns, tektin-4, indicated a significant effect on spermatozoa motility (P < 0.05) albeit non-significant on spermatozoa morphology. The bulls which showed pattern A had a higher percentage of spermatozoa motility than pattern E (P < 0.05) and not different from pattern C (P > 0.05). The statistical analysis also revealed that the presence of spot Z₁ had an effect on the percentage of spermatozoa motility (P < 0.01), whereas the presence of spot Z₂ and Z₃ had no effect (P > 0.05). The correlation coefficient between the relative protein content of spot Z₁ and the percentage of spermatozoa motility was 0.49. Our study demonstrates that the expression patterns of tektin-4 were a proxy for an effect on spermatozoa motility and consequently bull infertility. It may be that these protein patterns can be used as markers for improving bovine reproduction.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

The Interactions of Porcine and Ovine,Serum and Colostral Immunoglobulins with Staphylococcal Protein A

The purpose of these studies was to determine the proportion of each immunoglobulin class/subclass in blood and colostrum of the pig and sheep, which would bind to staphylococcal Protein A. The concentrations of porcine IgG, IgM, and IgA were determined for serum and colostral whey from five sows. Similar measurements were made on two fractions produced by elution of the sample through a Protein A-Sepharose column: fraction 1, immunoglobulins which did not bind to Protein A, and fraction 2, immunoglobulins which bound to Protein A. The concentrations of ovine IgG₁, IgG₂, IgM, and IgA were measured for serum and colostral whey from six ewes, and again similar measurements were made after elution of each ovine sample through Protein A-Sepharose. All classes/subclasses of porcine and ovine serum and colostral immunoglobulins bound to Protein A to some extent. More than 90% of IgG from both porcine colostral whey and serum bound to Protein A. Ovine IgG₁ from most ewes possessed a low affinity for Protein A whereas ovine IgG₂ generally possessed a high affinity; 100% of the IgG₂ in ovine colostral whey samples bound to Protein A. There was remarkable variation between individuals in the binding capacity of porcine IgM and each of the ovine immunoglobulins. For the ovine samples, in particular there were distinct differences between Protein A binding capacity of serum and colostral immunoglobulins of the same class/subclass.     

The effect of the F gene on male reproductive traits in Booroola × South Australian Merino rams

The effect of the Booroola (Bo) F gene on the reproductive development of Bo × South Australian Merino ($\text{1}\text{2}\text{Bo}$) rams was examined. Half Bo rams were offspring of FF (homozygous carrier), F+ (heterozygous carrier) or ++ (homozygous non-carrier) Bo sires, so that the expected frequency of $\text{1}\text{2}\text{Bo}$ rams carrying the gene should be approximately 1.0, 0.5 or 0.0, respectively. The genotype of each Bo sire was defined on the basis of the proportion of daughters with ? 3 ovulations. The reproductive development of the $\text{1}\text{2}\text{Bo}$ rams corresponding with each sire genotype was studied between 5 and 15 months of age. Traits recorded were: live weight, testicular volume, preputial adhesion score, semen volume, semen density, proportion of rams with ejaculates containing spermatozoa, proportion of rams that mounted and proportion of rams that served oestrous ewes. There were no significant differences among sire genotypes in the traits studied, except for preputial adhesion score (FF < F+ = ++, P < 0.05) and proportion of rams that mounted (FF = F+ < ++, P < 0.05) at 5 months of age. The relevance of these differences is not known but could result from sampling problems. It is concluded that (1) the F gene had no discernible role in male reproductive development between 5 and 15 months of age and (2) the traits examined did not allow a distinction between carrier and non-carrier rams of the F gene.     

The effect of oxygen on CO2 fixation by mesophyll protoplast extracts of C3 and C4 plants.

Oxygen inhibits CO₂ fixation by mesophyll protoplast extracts of the C₃ plants, $\text{Hordeum vulgare}$ and $\text{Triticum aestivum}$, but stimulates the pyruvate induced CO₂ fixation by mesophyll protoplast extracts of the C₄ plants $\text{Digitaria sanguinalis}$ and $\text{Urochloa panicoides}$. The former is reversed by increased levels of bicarbonate, whereas the latter effect is independent of bicarbonate concentration. The results are consistent with the proposal that oxygen inhibits C₃ photosynthesis by competing with CO₂ in the RuDP carboxylase/oxygenase system. The oxygen enhancement of C₄ mesophyll photosynthesis is proposed to be due to pseudocyclic electron flow supplying additional ATP for the CO₂ fixation process.     

Effect of different levels and sources of zinc supplementation on quantitative and qualitative semen attributes and serum testosterone level in crossbred cattle (Bos indicus x Bos taurus) bulls

An experiment was conducted on 16 crossbred bulls (about 2 years of age, 316.2+/-0.77 kg average body weight), divided into groups I, II, III and IV to study the effect of different levels of Zn supplementation from inorganic and organic sources on semen quality. The animals in the first 3 groups were supplemented with 0, 35 and 70 ppm Zn from Zn sulfate, respectively and the animals in-group IV were supplemented with 35 ppm Zn as Zn propionate. Semen collection and evaluation was done in the first month (to assess semen quality at the start of the experiment) and 7th, 8th and 9th month of experimental feeding to evaluate the effect of supplemental Zn on semen attributes. We gave 6 months for Zn feeding, so that 3 sperm cycles of spermatogenesis had passed and the collected semen reflected the complete effect of Zn supplementation. Six ejaculates from each bull were collected and evaluated for semen quantitative (ejaculate volume, sperm concentration and sperm number per ejaculate) and qualitative characteristics (semen pH, mass motility, individual motility, sperm livability percent and abnormal sperm percent, percent intact acrosome, bovine cervical mucus penetration test, hypo-osmotic sperm swelling test) and activity of seminal plasma enzymes i.e., alkaline phosphatase, acid phosphatase, GOT and GPT. Testosterone level in the blood serum of crossbred bulls was also estimated. Mean values of semen quantitative and qualitative characteristics at the start of the experiment were statistically non significant (P > 0.05) in all the crossbred cattle bulls, however, there were statistically significant differences among the bulls of different groups after 6 months of zinc supplementation. Mean ejaculate volume (mL) was 2.37, 4.70, 5.86 and 6.38, respectively in groups I to IV, indicating a statistically significant (P < 0.05) higher semen volume in Zn-supplemented groups as compared to the control group of bulls. Similarly, sperm concentration (million.mL(-1)), live sperm (%) and motility (%) were significantly (P < 0.01) higher in Zn-supplemented groups as compared to the control group. The results of BCMPT and HOSST revealed a significant improvement in sperm functional ability in all the groups supplemented with Zn as compared to the control group. The activity of alkaline and acid phosphatase in seminal plasma was significantly (P < 0.05) higher in the Zn-supplemented groups, whereas GOT and GPT activities in seminal plasma were significantly (P < 0.05) lower in the Zn propionate supplemented group as compared to the control group. Testosterone concentration (ng.mL(-1)) in blood serum was significantly higher in animals of groups III and IV, as compared to control group. It may be concluded that Zn supplementation either in the inorganic or organic form in the diet of crossbred bulls improved the qualitative and quantitative attributes of semen; however, the number of sperm per ejaculate, mass motility and semen fertility test like bovine cervical mucus penetration was significantly higher in bulls given Zn in an organic form (Zn propionate) as compared to an inorganic form (Zn sulfate).