Associação entre proteínas do plasma seminal, motilidade e viabilidade espermática em coelhos submetidos a doping genético / Association among seminal plasma proteins, sperm motility and sperm viability in rabbits submitted to gene doping

Neste trabalho foi estudada a correlação entre o perfil proteico do plasma seminal e a motilidade e viabilidade espermática em coelhos submetidos ao tratamento com vetores de expressão contendo o gene da eritropoetina (EPO) e com EPO recombinante humana. Foram identificadas, em coelhos submetidos ao tratamento com vetor de DNA contendo o gene da EPO, duas bandas proteicas associadas a alterações na motilidade espermática - 48kDa à baixa motilidade (P<0,05) e 18kDa à alta motilidade (P<0,05) - e esse fator foi associado a maior viabilidade espermática (P<0,05). Em coelhos submetidos ao tratamento com EPO recombinante, um fator proteico, 63kDa, associou-se à alta motilidade espermática (P<0,05), enquanto dois, 26 e 40kDa, foram associados à alta viabilidade espermática (P<0,05). Esses resultados sugerem que o doping genético pode ocasionar mudanças no perfil proteico do plasma seminal, provocando alterações na motilidade e viabilidade espermática.

In this study the correlation between seminal plasma protein profile and the sperm motility and sperm viability in rabbits submitted to treatment with an expression vector containing EPO gene and with human recombinant EPO was evaluated. In rabbits submitted to treatment with EPO expression vector, two protein bands were associated to sperm motility - 48kDa associated to low motility (P<0.05) and 18kDa to high motility (P<0.05) - and this protein band was also associated to high sperm viability (P<0.05). In rabbits submitted to treatment with human recombinant EPO, a protein factor, 63kDa, was associated to high sperm motility (P<0.05) while two protein factors, 26 and 40kDa, were associated to high sperm viability (P<0.05). These results suggest that gene doping leads to changes in rabbit seminal plasma protein, altering sperm motility and sperm viability.

Erythropoietin non-viral gene therapy does not affect motility, viability, morphology or concentration of rabbit sperm.

Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker.

The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P<0.01) and MIn (45.8%; P<0.05) of the oocytes (N=59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P<0.05), whereas MIn remained compromised in this group (N=67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.

NanoSMGT: transfection of exogenous DNA on sex-sorted bovine sperm using nanopolymer.

The objective was to introduce exogenous DNA into commercially sex-sorted bovine sperm using nanopolymer for transfection. In the first experiment, the optimal concentration and ratio of linear-to-circular plasmid was determined for NanoSMGT in unsorted sperm. A second experiment was conducted to transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake occurred in a dose-dependent manner (P < 0.05). The optimal amount of DNA was 10 µg/10(6) cells. The ratios of linear-to-circular plasmid do not influence the uptake by unsorted sperm cells and none of the tested treatments affected sperm motility and viability. Commercially sex-sorted bovine sperm were able to uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted sperm had decreased DNA uptake in comparison to unsorted sperm (P < 0.05). Neither sperm motility nor viability were affected by nanotransfection. In conclusion, nanopolymer efficiently introduced exogenous DNA into commercially sex-sorted bovine sperm; we inferred that these sperm could be used for production of embryos of the desired sex, a technique named NanoSMGT.

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus / Cloning and evaluation of sbGnRH gene expression in juvenile and adult males of Brazilian flounder Paralichthys orbignyanus

Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

In vitro penetration of swine oocytes by homologous spermatozoa: Distinct systems for gamete's co-incubation and oocyte's cryopreservation.

In vitro penetration (IVP) of swine oocytes by homologous spermatozoa was evaluated in two experiments using four boars as semen donors. In experiment 1, the IVP rate and the number of penetrating spermatozoa (PSP) were compared using three co-incubation systems for vitrified oocytes and fresh sperm: (1) 35mL petri dishes in a CO(2) incubator, (2) 35mL petri dishes in bags (submarine system) and (3) glass flasks partially submerged in water bath with the same gas mixture used for the bag system. Mean PSP was 8.2+/-10.1 and the IVP rate was 90.5%. The PSP differed across all systems (P=0.0006): 15.5+/-0.5 for flasks, 6.3+/-0.4 for CO(2), and 3.9+/-0.4 for bags. The IVP rate for flasks (95.0%) was greater (P=0.01) than for CO(2) and bags (90.8% and 85.0%, respectively), but it did not differ between flasks and CO(2) for three boars (P>0.05). In experiment 2, co-incubation was done as described for glass flasks in experiment 1. The IVP rate and PSP were compared for cryopreserved oocytes: either vitrified in open pulled straws (OPS), or frozen in cryotubes. Mean PSP was 5.4+/-6.5 and IVP rate was 89.6%. Both PSP and IVP rate were greater (P<0.0001) for oocytes frozen in cryotubes (7.0+/-0.3% and 95.8%, respectively) than those frozen in OPS (3.7+/-0.3% and 83.4%, respectively), with no differences found for three boars (P>0.05). In summary, successful IVP of swine oocytes by homologous spermatozoa can be achieved using gametes incubated in glass flasks and oocytes frozen in cryotubes.

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay.

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Although isolated IPVL can be stored for up to 24h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean+/-SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0+/-17.7 holes/mm(2) IPVL and 159.5+/-17.7 holes/mm(2) IPVL, respectively, P>0.05; n=123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.

Effect of low density lipoprotein on the quality of cryopreserved dog semen.

Egg yolk is included in extenders for semen cryopreservation due to its protective effect against cold shock, which is attributed to the presence of low density lipoprotein (LDL). This study evaluates how semen quality is affected by using LDL as a replacement for egg yolk in extenders for cooled and frozen dog semen. In Experiment 1, semen was extended in TRIS-glucose at 5 degrees C, in four treatments: 20% egg yolk (T1); 6% (T2); 8% (T3); and 10% LDL (T4). Sperm motility and membrane integrity after 24, 48, 72 and 96 h and the 50% conservation rate of motile spermatozoa (50 M) were evaluated. The 50 M was less for T1 than for the other treatments (P<0.01), but T2-T4 did not differ (P>0.05). In Experiment 2, glycerol at 10% was included in the freezing extender, in treatments similar to those from Experiment 1. Sperm motility and membrane integrity did not differ for T2, T3 and T4 at any period in Experiment 1 and after thawing in Experiment 2 (P>0.05), but were greater for all LDL treatments than for T1 (P<0.01), in both experiments. Thus, LDL can replace egg yolk in the composition of the TRIS-glucose extender for cooled or frozen dog semen.

Fator do plasma seminal associado à integridade de membrana de espermatozóides suínos pós-descongelamento / Seminal plasma factor associated to post-thawing swine sperm membrane integrity

Neste estudo, identificaram-se polipeptídeos associados à integridade da membrana plasmática (IMP) de espermatozóides suínos após o processo de congelamento/descongelamento. Por meio do perfil protéico do plasma seminal em SDS-PAGE, observou-se a presença de nove bandas polipeptídicas com pesos moleculares que variaram de 11,97 a 122,52kDa. Detectou-se que uma banda de 26,58kDa esteve associada à baixa IMP (<55 por cento). Não foi verificada associação entre as outras bandas e a IMP. Conclui-se que o fator polipeptídico de 26,58kDa está associado à baixa integridade da membrana plasmática do espermatozóide suíno após o congelamento/descongelamento.

Polypeptides associate to membrane integrity (MI) of swine spermatozoa submitted to freezing and thawing were identified. The protein profile of seminal plasma analyzed by SDS-PAGE allowed the identification of nine polypeptide bands with molecular weight ranging from 11.97 to 122.52kDa. One 26.58kDa band was associated with reduced MI (<55 percent). No associations among other bands and MI were observed. The 26.58kDa factor is associated with reduction of membrane integrity of swine spermatozoa after freezing and thawing.

Evaluation of amides and centrifugation temperature in boar semen cryopreservation.

Two experiments were conducted to evaluate the use of amides as cryoprotectants and two centrifugation temperatures (15 or 24 degrees C) in boar semen cryopreservation protocols. Semen was diluted in BTS, cooled centrifuged, added to cooling extenders, followed by the addition of various cryoprotectants. In experiment 1, mean (+/-S.E.M.) sperm motility for 5% dimethylformamide (DMF; 50.6+/-1.9%) and 5% dimethylacetamide (DMA; 53.8+/-1.7%) were superior (P<0.05) to 5% methylformamide (MF; 43.2+/-2.4%) and 3% glycerol (GLY; 38.1+/-2.3%), with no significant difference between MF and GLY. Sperm membrane integrity was higher (P<0.05) for DMA than for MF or GLY (50.9+/-1.9, 43.3+/-2.5, and 34.5+/-2.8%, respectively). Sperm membrane integrity was higher in DMF (47.9+/-2.1%) than in glycerol (34.5+/-2.8%, P<0.05), but was similar to other treatments (P>0.05). In experiment 2, we tested MF, DMF, and DMA at 3, 5, and 7%. Sperm motility and membrane integrity were higher for 5% DMA (53.8+/-1.7 and 50.9+/-1.9%) and 5% DMF (50.6+/-1.9 and 47.9+/-2.1%), in comparison with 7% DMF and all MF concentrations (P<0.05). For sperm motility and membrane integrity, 5% DMA exceeded (P<0.05) 3% DM, with greater membrane integrity than 3% DMF (P<0.05). In both experiments, sperm motility and membrane integrity were superior at 15 degrees C versus 24 degrees C (P<0.05), with no interaction between centrifugation temperature and treatments (P>0.05). In conclusion, boar semen was successfully cryopreserved by replacement of glycerol with amides (especially 5% DMA) and centrifugation at 15 degrees C, with benefits for post-thaw sperm motility and membrane integrity.

Reproductive performance of early-weaned female swine according to their estrus profile and frequency of artificial insemination.

We determined the estrus profile (weaning-to-estrus interval (WEI), estrus duration (ED), and frequency of estrus per detection period) in 184 female swine and estimated the effect of the WEI, ED and frequency of artificial insemination (AI) on farrowing rate (FR) and litter size. Estrus detection was done at 8:30 a.m. and 5:00 p.m. The WEI was categorized as short (<100 h), medium (100-120 h) and long (>120 h). The ED was categorized as short (<60 h), medium (60-72 h) and long (>72 h). Mean lactation length was 14.6 days, mean WEI was 124.5 h and mean ED was 69 h. In each weaning group, females received either one or two AI, following a breeding schedule based on the estrus profile. In single-mated females, Al was performed 36 h after the beginning of estrus. In double-mated females, the first AI was done 24 h after the beginning of estrus and the second AI occurred 12 h later. The period of estrus detection had no effect (P > 0.05) on WEI, ED, FR, total born (TB) and live born litter size (LB). Mean FR was 82.6%, mean TB was 10.0% and mean LB was 9.2%. Mean ED was shorter (P < 0.03) for females having medium and long WEI (67.0 and 65.4 h, respectively) than for those having short WEI (72.2 h). A linear regression analysis identified a weak (R2 = 0.02) but significant negative association between ED and WEI (P = 0.05). The WEI did not influence FR (P > 0.05). Total litter size for females having short WEI (9.4) was lower (P < 0.03) than for those having long WEI (10.4). Also, LB for females having medium and long WEI (9.7-9.8) was higher (P < 0.05) than for those having short WEI (8.7). AI frequency had no effect on FR (P > 0.05). TB and LB litter size were lower (P < 0.05) for single-mated females (9.6 and 9.0, respectively) than for double-mated females (10.7 and 9.6, respectively). Double Al was associated with higher subsequent litter size. However, breeding schedules based only on estrus profile may not be precise in determining ideal breeding time, since females having short WEI had the longest ED and produced the lowest litter size.

Influence of equine chorionic gonadotropin on weaning-to-estrus interval and estrus duration in early-weaned, primiparous, female swine.

The weaning-to-estrus interval (WEI) influences the total nonproductive days (NPD) accumulated by the breeding herd and affects herd productivity. Short lactation lengths (LL) are commonly followed by prolonged WEI, which are also associated with short estrus duration (ED). Equine chorionic gonadotropin treatment is a tool that has been used to reduce WEI, especially for low-parity females. The objectives for this study were to evaluate the effect of LL on the association between WEI and ED and to estimate the effects of postweaning eCG administration on WEI and ED for early-weaned females. Two treatments (TREAT) consisting of 750 IU of eCG (n = 96) or control (n = 77) were applied 1 d after weaning to first-parity, weaned females. The study was conducted on a commercial farm having a target LL of 18 d. Estrus detection was conducted three times daily, and estrus duration was determined as the interval between the first and the last positive response to back pressure. Analyses of variance were conducted to estimate the effects of LL and TREAT on WEI and the effects of TREAT and WEI on estrus duration. Mean LL was 17.9+/-1.7 d, mean WEI was 106.6+/-29.2 h, and mean estrus duration was 55.9+/-15.5 h. Even though the frequency of short WEI tended to increase with longer LL, mean WEI was shortest for females weaned after 18 d and longest for those weaned after 20 d (P<.05). The WEI for females receiving eCG (98.7+/-2.7 h) was shorter (P = .0001) than that for control females (121.5+/-3.3 h). The WEI was also affected by a LL x TREAT interaction (P = .0014), indicating that the interval was longer (P<.05) for control females weaned after 17 and 20+ d than for other females. The LL and TREAT did not affect estrus duration (P = .20 and P = .157, respectively). However, estrus duration was reduced as the WEI increased (P = .0001), and it was also influenced by a WEI x TREAT interaction (P = .024). A linear regression model estimated that the association between WEI and estrus duration was stronger in the eCG group than in the control group (R2 = .51 and .15, respectively; both P<.001). In conclusion, the use of eCG postweaning was associated with more precise prediction of estrus duration as a function of the WEI and allows optimization of breeding management in early-weaned, primiparous females.

Effect of equine chorionic gonadotropin on weaning-to-first service interval and litter size of female swine.

We evaluated the effect of PMSG on the weaning-to-first service interval, total litter size and born alive litter size in swine. Four doses of PMSG (0, 500, 750 and 1,000 IU) were administered intramuscularly after weaning to sows at 3 different farms, grouped by parities (1, 2 and 3 or higher) and 2 distinct time periods. The associations among main effects and response variables were assessed by analysis of variance. Polynomial orthogonal terms were used to adjust the estimates of weaning-to-first service interval, total litter size and born alive litter size for the interaction effect of parity and PMSG treatment. The weaning-to-first service interval did not differ across periods and farms (P>0.05), although the interval was shorter (P<0.05) for Parity 3+ sows (4.97 d) than for Parity 1 sows (5.29 d), with no other differences in intervals observed across parities (P>0.05). Time period did not influence litter size (P>0.05), but there were differences in litter size across farms (P<0.05). Both litter size traits were lower for Parity 1 sows than for higher parity sows (P<0.05), but there were no differences in litter size between Parity 2 and 3+ sows (P>0.05). Litter size increased with PMSG dose in both Parities 1 and 2 (P<0.05), but not in Parity 3+ (P>0.05). A significant quadratic effect (P<0.05) of PMSG treatment in weaning-to-first service interval was observed for both Parity 1 and 2 sows, with the shortest intervals occurring with the 750 IU dose for Parity 1 sows. Administration of PMSG after weaning was associated with a shortened weaning-to-first service interval in Parity 1 sows and increased litter size in Parity 1 and 2 sows.

Comparison of sex ratio and cell number of IVM-IVF bovine blastocysts co-cultured with bovine oviduct epithelial cells or with Vero cells.

The influence of 2 co-culture systems (BOEC and Vero cells) on the development rates, quality grades and sex ratios of IVM-IVF bovine embryos were studied. Zygotes obtained after IVF were co-cultured in each co-culture system for 7 and 8 d (Day 0 = day of insemination) in B2 medium. No effect of the co-culture system was observed on development rates measured on Days 7 and 8. However, Vero cell co-culture had a positive influence on embryo quality. Irrespective of their sex, embryos produced on Vero cells showed higher cells number than those co-cultured on BOEC (103.4 +/- 3.8 and 97 +/- 8.12 for BOEC vs 113.7 +/- 3.5 and 114 +/- 5.9 for Vero cells at Days 7 and 8, respectively; P < 0.05). The percentage of male embryos was increased in the two co-culture systems (60.7% males for BOEC; P < 0.05 vs 63% males for Vero cells; P < 0.01) on Day 7. In both co-culture systems the increase in the percentage of males was more obvious for embryos reaching the most advanced stage (expanded blastocysts). The results show that Vero cells improved the quality grade of bovine embryos produced in vitro, and thus are recommended for use as a safe co-culture system that does not contain pathogens.

Effects of zeranol on reproduction in beef bulls: luteinizing hormone, follicle-stimulating hormone, and testosterone secretion in response to gonadotropin-releasing hormone and human chorionic gonadotropin.

Effects of zeranol on the maturation of the adenohypophyseal-gonadal axis were studied in beef bulls. Calves were implanted with 36 mg of zeranol at 3-month intervals from birth through 6 months of age (group 2, n = 10) or were not treated (control group 1, n = 10). After 9 months, group-2 calves were given implants of 36 mg of zeranol at 3-month intervals through 18 months of age (group 2B, n = 5) or were not reimplanted (group 2A, n = 5). Areas under the curves outlined by concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone for 6 hours after the administration of 100 micrograms of gonadotropin-releasing hormone (GnRH) were calculated. Gonadotropin-releasing hormone was administered at 3-month intervals from 1.5 through 19.5 months of age. Areas under the curves for concentrations of testosterone for 4 hours after the administration of 10,000 IU of human chorionic gonadotropin (HCG) at 4.5, 7.5, and 10.5 months or 1,000 IU at 13.5 and 16.5 months of age also were calculated. The amount of FSH released was greater (P less than 0.05) for group-2 than for group-1 calves at 4.5 and 7.5 months of age. The amount of FSH released in groups 2A and 2B tended (P less than 0.10) to be greater than that for group 1. Significant differences between groups 2A and 2B were not observed. The amount of LH released at 7.5 months of age was less for groups 1 and 2 than that at earlier ages, and the decrease was greater (P less than 0.05) for group 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of zeranol on reproduction in beef bulls: scrotal circumference, serving ability, semen characteristics, and pathologic changes of the reproductive organs.

Effects of zeranol on scrotal circumference, serving ability, semen characteristics, and postmortem measurements of the genital organs were determined in beef bulls from 9 to 20 months of age. Group 1 (n = 5) served as a nonimplanted control group. Group 2A (n = 5) was implanted with 36 mg of zeranol at birth and at 3 and 6 months of age. Group 2B (n = 5) was implanted with 36 mg of zeranol every 3 months from birth through 18 months of age. Scrotal circumference was adversely affected by zeranol in groups 2A and 2B, but values approached those of group 1 with increasing age. Serving ability was also affected adversely but tended to recover with increasing age. Semen quality was low in groups 2A and 2B and did not improve with increasing age. There was no difference in testicular weight, vesicular gland weight, and penis length among groups when bulls were slaughtered at 20 months of age. Epididymal weight was greater in group-2B bulls and was most likely a consequence of epididymal lesions. Histologic examination of the genital organs revealed that zeranol induced adenomyosis and sperm granulomas in the caudae epididymidis and markedly altered the structure of the sexual accessory glands of bulls in groups 2A and 2B. Alterations in the vesicular glands were characterized by reduced alveolar development and an increase in connective tissue. Low epithelium associated with focal areas of squamous metaplasia were common in the prostate of groups 2A and 2B bulls. Lesions in the bulbourethral glands were characterized by low glandular epithelium, focal areas of squamous metaplasia, cystic collecting ducts, and an increase in connective tissue. Groups 2A and 2B had more abnormal seminiferous tubules than did group 1. Lesions in groups 2A and 2B may have been direct effects of zeranol or may have resulted from reduced testosterone secretion.