Plasma urea levels on reproductive parameters of wool-less rams (Ovis aries, LINNAEUS, 1758)

Nível plasmático de uréia sobre parâmetros reprodutivos de machos ovinos deslanados (Ovis aries, LINNAEUS, 1758)

Plasma urea levels on reproductive parameters of wool-less rams (Ovis aries, LINNAEUS, 1758)^*

Nível plasmático de uréia sobre parâmetros reprodutivos de machos ovinos deslanados (Ovis aries, LINNAEUS, 1758)

Carmen Neusa Martins CORTADA¹; Carlos de Sousa LUCCI²; Rodrigo Alonso Forero GONZALEZ¹; Renato VALENTIN³; Carla Balzano de MATTOS⁴

CORRESPONDENCE TO:

Carmen Neusa Martins Cortada

Departamento de Reprodução Animal

Faculdade de Medicina Veterinária e Zootecnia da USP

Av. Prof. Dr. Orlando Marques de Paiva, 87

Cidade Universitária Armando de Salles Oliveira

05508-000 – São Paulo – SP

e-mail: [e-mail]

SUMMARY

The effect of increased plasma urea levels on reproductive parameters of Santa Ines Brazilian breed rams (Ovis aries) was determined during two phases, 60 days in length each. During the first phase, 18 animals divided into three blocks according to scrotal circumference were submitted to the following treatments: diet A₁- with nitrogen levels according to ARC (1980); diet B_{1 -} nitrogen levels 133% higher than A₁;and diet C₁ - nitrogen levels 166% higher than A₁. During the second phase, 12 animals divided into two groups received diet A₂- with nitrogen levels according to ARC (1980), and diet B₂ - nitrogen levels 200% higher than diet A₂, respectively_. The increase in protein equivalents was obtained by adding 20, 40 and 60 g of feed grade urea to treatments B₁, C₁ and B₂, respectively. Semen was collected daily using an artificial vagina and analyzed twice a week for progressive motility; vigor, whirling, density, pH, concentration, sperm morphology, ejaculated volume and total sperm per ejaculate. Urea concentration was determined weekly in semen and plasma. Urea level increased in plasma and semen (p < 0.0001) during both phases but did not affect the parameters studied.

UNITERMS: Urea; Semen; Reproduction; Rams; Feed Ration.

INTRODUCTION

The achievement of high levels of fertility and prolificacy in sheep flocks relies not only upon the female members but also upon their male consorts¹.

Diets with high levels of crude protein, nitrogen compounds or lack of a ruminal substrate for complete transformation of ammonia into bacterial protein may increase rumen and consequently plasma urea concentration. Plasmatic urea can reach the seminiferous tubules^26,34 and the presence of testicular transporters³⁹ for urea suggests that it may play a role in spermatogenesis³⁹, despite increased plasma urea concentration in humans due to renal insufficiency^{17,18,19,20,24,25,28,31}, andin dairy cows^{7,8,9,12,13,14,15,21,22} due to high levels of nitrogen compounds in the diet, are considered to predispose to reduced fertility. Impaired reproduction in bulls^6,10 reportedwith increased dietary nitrogen compounds was not always confirmed neither in bulls^4,32,33,38 nor in rams^29,30,38.

The present study investigated the effect of increased plasma urea levels on the reproductive parameters of rams.

MATERIAL AND METHOD

Hybrid wool-less Santa Ines rams aged 11 to 18 months and weighing 33.1 to 41.4 kg (live weight) individually pen housed indoors were submitted to daily sperm collection with an artificial vagina kept at 46ºC, using females as manikins, from May to October 1996 at the Center of Biotechnology in Animal Reproduction, Pirassununga (BR), located at 21º59’ South latitude and 47º33’ West longitude of Greenwich. Ambient temperature ranged from 12.4ºC to 25.0ºC and daytime duration from 10 h and 56 min to 12 h and 44 min, during the experimental period.

The treatments consisted of isoenergetic diets containing different nitrogen levels, in two different phases, as follows.

During phase 1 (May to July), 18 animals were used in a generalized randomized block design¹⁶ with three treatments and three blocks and treatment replicates within blocks. The animals were assigned to the different blocks according to scrotal circumference. Treatments were structured with three increasing levels of dietary nitrogen: diet A₁- ration containing nitrogen levels according to², or 0 g of urea; diet B₁ - a 133% increase in nitrogen compared to diet A_1, or 20 g urea, gradually increased and diet C₁ - a 166% increase in nitrogen compared to diet A₁, or 40 g of urea, gradually increased. Cornmeal was used on all diets, serving as a vehicle for urea in diets B₁ and C₁.

During phase 2 (August to October), 12 animals previously assigned to treatments A₁ and C₁ (phase 1) were kept in phase 2 and renamed as treatments A₂ and B₂. The animals previously assigned to treatment B₁ were discarded. A fully randomized design was used¹⁶, with two treatments and six replicates per treatment. The treatments consisted of diet A₂ - ration with a nitrogen level according to², or 0 g of urea and diet B₂, a 200% increase in protein equivalents compared to diet A₂, or 60 g urea, gradually increased from diet C₁. Cornmeal was added to all diets, serving as vehicle for urea in diet B₂.

The composition of all the rations is shown in Tab. 1. The parameters started to be analyzed only after the desired amount was reached in each ration.

Table 1

Composition of daily amount ingredients and nutrients during phases 1 and 2, Pirassununga, 1996.

a Composition (% of dry matter) of the pellet mixture: Coast cross hay (IFN 1-00-716). 30.0%; Soybean meal (IFN 5-04-604), 7.0%; Cornmeal (IFN 4-21-018), 61.5%; Mineral supplement, 1.5%.

The following seminal parameters were analyzed twice a week: progressive straight motility²⁷, vigor²⁷, whirling²⁷, density¹¹, volume¹¹, sperm concentration²⁷, total number of ejaculated spermatozoa¹¹, pH, measured with a portable digital potentiometer (CORNING^® - EUA) soon after collection, and sperm morphology^3,5, using an interference contrast microscope (JENAVAL^® Germany). Semen and blood plasma aliquots were collected weekly and stored at –20ºC for later urea determination by the colorimetric enzymatic method using a commercial kit (CELM^® - BR). For semen urea determination, the aliquot was first submitted to deproteination¹¹.

Scrotal circumference was measured (cm) at 15 days intervals using a specific tape (Lane Manufacturing Inc.^® EUA) and testis and epidydimis weight were determined after surgical removal of the gonads using an analytical scale, at the end of phase two.

Data was analyzed statistically using the Statistical Analysis System software³⁶ after residue normality tested by the Shapiro-Wilk test (Proc UNIVARIATE) and variances compared by the F test. Phase 1 data for plasma and seminal urea, seminal parameters and scrotal circumference were submitted to analysis of variance by the GLM procedure (PROC GLM) and means separation was achieved by orthogonal contrasts. The effects of treatment for morphological sperm alterations were determined by non-parametric rank analysis using the Kruskal-Wallis test. Phase 2 data for plasma and seminal urea, seminal parameters, scrotal circumference, testis and epidydimis weight were submitted to analysis by the t-test (PROC TTEST). For data concerning morphological sperm alterations, the effect of treatment was determined by non-parametric rank analysis using the Wilcoxon 2-sample test. The level of significance was set at 5% in all analyses.

RESULTS, DISCUSSION AND CONCLUSION

There was an increase (p < 0.0001) in plasma and semen urea concentration (Tab. 2) that had no effect on the semen parameters analyzed (Tabs. 3 and 4), as also described by Thompson et. al.³⁸, who detected no difference in semen quality or fertility of bulls and rams. The urea increase also had no effect on testis and epidydimis weight and on scrotal circumference (Tab. 5) according to Rocha et. al.³². However, these results were in contrast to those reported by Branton et. al.⁶ and Castillo et. al.¹⁰, who observed a reduction of these parameters in bulls with increased amount of dietary protein. The results were also in contrast to remarks in which were observed gonadal degeneration and infertility, with reduced sperm production and loss of libido, attributed to the increase in plasma urea levels^{17,18,19,20,24,28,31} in human patients with renal insufficiency. The lack of effects in these rams’ semen parameters, despite the urea increase in plasma and semen urea levels may be associated to levels not high enough to become harmful, since plasma urea levels remained below the broad range considered to be normal in rams by different authors, e.g., from 17 to 42²³ mg/dl or 43 to 75 mg/dl^35,37.

Table 2

Urea concentration (mg/dl) in plasma and semen (mean ± sem), coefficient of variation (C.V.), statistical probability, regression equation and coefficient of determination (R2), Pirassununga, 1996.

a SEM, Standard error of the mean; b P values for linear regression and deviations; c P values for the t-test.

Table 3 Semen parameters (mean ± sema), coefficient of variation (C.V.), statistical probability, of rams fed with increasing nitrogen levels, Pirassununga, 1996. aSEM, Standard error of the mean; b P values for linear regression and deviations; c P values for the t-test.

Table 3

Semen parameters (mean ± sema), coefficient of variation (C.V.), statistical probability, of rams fed with increasing nitrogen levels, Pirassununga, 1996.

aSEM, Standard error of the mean; b P values for linear regression and deviations; c P values for the t-test.

Table 4 Sperm morphology alterations (mean ± sema), coefficient of variation (C.V.) and statistical probability of rams fed with increasing nitrogen levels, Pirassununga, 1996. aSEM, Standard error of the mean; b P values for Kruskall Wallis rank test; c P values for Wilcoxon 2 - sample rank test.

Table 4

Sperm morphology alterations (mean ± sema), coefficient of variation (C.V.) and statistical probability of rams fed with increasing nitrogen levels, Pirassununga, 1996.

aSEM, Standard error of the mean; b P values for Kruskall Wallis rank test; c P values for Wilcoxon 2 - sample rank test.

Table 5

Testis plus epidydimis weight (mean ± sema) coefficient of variation (C.V.) and statistical probability, Pirassununga , 1996.

aSEM, Standard error of the mean; b P values for the t - test.

RESUMO

Estudou-se o efeito de níveis aumentados de uréia plasmática sobre parâmetros reprodutivos de machos ovinos (Ovis aries) deslanados da raça Santa Inês, em duas fases de 60 dias cada. Durante a primeira fase, 18 animais divididos em três blocos de acordo com perímetro escrotal foram submetidos aos seguintes tratamentos: dieta A₁ – com níveis de nitrogênio de acordo com ARC(1980); dieta B₁ – com níveis de nitrogênio 133% maiores que dieta A₁; e dieta C₁ – níveis de nitrogênio 166% maiores que dieta A₁. Durante a segunda fase, 12 animais divididos em dois grupos receberam as dietas: A₂ – com níveis de nitrogênio de acordo com ARC (1980) e dieta B₂ – níveis de nitrogênio 200% maiores que dieta A₂. O aumento no equivalente protéico foi obtido com adição de 20, 40 e 60 g de uréia aos tratamentos B₁, C₁ e B₂, respectivamente. Foi coletado sêmen diariamente com vagina artificial e duas vezes por semana foram analisados: motilidade retilínea e progressiva, vigor, turbilhonamento, densidade, pH, concentração, morfologia espermática, volume ejaculado e total de espermatozóides por ejaculado. A concentração de uréia no plasma e no sêmen foi analisada semanalmente. O aumento (p < 0,0001) de uréia no sêmen e no plasma durante ambas as fases não afetou os parâmetros estudados.

UNITERMOS: Uréia; Sêmen; Reprodução; Carneiros; Rações.

Received: 22/03/2000

Accepted: 30/11/2000

• ¹
  - ALKASS, J.E.; BRYANT, M.J. Some effects of level of feeding and body condition upon sperm production and gonadotropin concentrations in the ram. Animal Production, v.34, n.3, p.265-77, 1982.
• ²
  - ARC – AGRICULTURAL RESEARCH COUNCIL. The nutrient requirements of ruminants livestock Farnham Royal : CAB, 1980. 351p.
• ³
  - BARTH, A.D.; OKO, R.J. Abnormal morphology of bovine spermatozoa 1.ed. Ames : Iowa State University Press. Ames, 1989. 285p.
• ⁴
  - BHOSREKAR, M.R.; JOSHI, A.G.; SABNE, K.S.; NAGENDRA, G.; WAGHMARE, B.S.; PUROHIT, J.R. Semen quality of cross-bred bulls fed on three different levels of digestible crude protein. Indian Journal of Dairy Science, v.39, n.4, p.215-20, 1986.
• ⁵
  - BLOM, E. Ultrastrukturen af nogle karakteristiske spermiedefekter og forslag til et nyt klassificerings-system for tyrens spermiogram. [The ultrastructure of some characteristic sperm defects and a proposal for new classification of bull spermogram]. Nordisk. Veterinaermedicin, v.25, n.7, p.383-91, 1973.
• ⁶
  - BRANTON, C.; BRANTON, R.W.; SALISBURY, G.W. Total digestible nutrients and protein levels for diary bulls used in artificial breeding. Journal of Dairy Science, v.XXX, n.12, p.1003-13, 1947.
• ⁷
  - BUTLER, W.R.; CALAMAN, J.J.; BEAM, S.W. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. Journal of Animal Science, v.74, n.6, p.858-65, 1996.
• ⁸
  - CALFIELD, R.W.; SNIFFEN, C.D.; BUTHER, W.R. Effect of excess degradable protein on postpartum reproduction and energy balance in dairy cattle. Journal of Dairy Science, v.73, n.9, p.2342-9, 1990.
• ⁹
  - CARROL, D.J.; BARTON, B.A.; ANDERSON, G.W.; GRINDLE, B.P. Influence of dietary crude protein intake on urea-nitrogen and ammonia concentration of plasma, ruminal and vaginal fluids of diary cows. Journal of Dairy Science, v.70, p.117, 1987. Supplement 1.
• ¹⁰
  - CASTILLO, E.; TIZOL, G.; ALVAREZ, J.L.; PEREZ, M.; BAEZ, R. Reduction of the protein concentrate level in the ration for Holstein sires. I. Effect of semen quality. Cuban Journal Agricultural Science, v.21, n.3, p.247-50, 1987.
• ¹¹
  - CORTADA, C.N.M. Efeito de níveis crescentes de nitrogênio não protéico na dieta sobre níveis de uréia plasmáticos, parâmetros reprodutivos e citogenéticos de ovinos (Ovis aries, LINNAEUS, 1758) deslanados São Paulo, 1998. 82p. Tese (Doutorado) – Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo.
• ¹²
  - ELROD, C.C.; AMBURGH, M.; VAN BUTLER, W.R. Alterations of pH in response to increased dietary protein in cattle are unique to the uterus. Journal of Animal Science, v.71, n.3, p.702-6, 1993.
• ¹³
  - ELROD, C.C.; BUTLER, W.R. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. Journal of Animal Science, v.71, n.3, p.694-701, 1993.
• ¹⁴
  - FERGUSON, J.D.; BLANCHARD, T.; GALLIGAN, D.T.; HOSHALL, D.C.; CHALUPA, W. Infertility in dairy cattle fed a high percentage of protein degradable in the rumen. Journal American Veterinary Association, v.192, n.5, p.659-62, 1988.
• ¹⁵
  - FERGUSON, J.D.; GALLIGAN, D.T.; BLANCHARD, T.; MATTHEW, R. Serum urea nitrogen and conception rate: the usefulness of test information. Journal of Dairy Science, v.76, n.12, p.3742-6, 1993.
• ¹⁶
  - GOMES, F.P. Curso de estatística experimental 9.ed. Piracicaba : Escola Superior de Agricultura "Luiz de Queiroz", SP, 1987. 467p.
• ¹⁷
  - HANDELSMAN, D.J. Hypothalamic-pituitary gonadal disfunction in renal failure, dialysis and renal transplantation. Endocrine Reviews, v.6, n.2, p.151-82, 1985.
• ¹⁸
  - HANDELSMAN, D.J.; SPALIVIERO, J.A.; TURTLE, J.R. Testicular function in experimental uremia. Endocrinology, v.117, n.5, p.1974-83, 1985.
• ¹⁹
  - HANDELSMAN, D.J.; SPALIVIERO, J.A.; TURTLE, J.R. Hypothalamic-pituitary function in experimental uremic hypogonadism. Endocrinology, v.117, n.5, p.1984-95, 1985.
• ²⁰
  - HOLDSWORTH, S.R.; DE KRESTSER, D.M.; ATKINS, R.C. A comparison of hemocialysis and transplantation in reversing the uremic disturbance of male reproductive function. Clinical Nephrology, v.10, n.4, p.146-50, 1978.
• ²¹
  - JORDAN, E.R.; SWANSON, L.V. Serum progesterone and luteinizing hormone in dairy cattle fed levels of crude protein. Journal of Dairy Science, v.48, n.5, p.1154-8, 1979.
• ²²
  - JORDAN, E.R.; SWANSON, L.V. Effect of crude protein on reproductive efficiency, serum total protein, and albumin in the high-producing diary cow. Journal of Dairy Science, v.62, n.1, p.58-63, 1979.
• ²³
  - KANEKO, J.J. Clinical biochemistry of domestic animals. 3.ed. New York : Academic Press, 1980. 832p.
• ²⁴
  - LIM, V.S. Reproductive function in patients with renal insufficiency. American Journal of Kidney Diseases, v.9, n.4, p.363-7, 1987.
• ²⁵
  - LIM, V.S.; FANG, V.S. Restoration of plasma testosterone levels in uremic men with clomiphene citrate. Journal of Clinical Endocrinology and Metabolism, v.43, n.6, p.1370-7, 1976.
• ²⁶
  - MANN, T.; MANN, C.L. Male reproductive function and semen Themes and trends in physiology, biochemistry and investigative andrology. Berlin : Spring-Verlag, 1981. 495p.
• ²⁷
  - MIES FILHO, A. Reprodução dos animais e inseminação artificial 5.ed. Porto Alegre : Sulina, 1987. 750p.
• ²⁸
  - NETTO JR, N.R.; PECORARO, G.; SABBAGA, E.; GÓES, G.M. Spermatogenesis before and after renal transplant. International Journal of Fertility, v.25, n.2, p.131-3, 1980.
• ²⁹
  - OKÓLSKI, A. Effect of high protein levels on the reproductive ability in rams. In: INTERNATIONAL CONGRESS ON ANIMAL REPRODUCTION AND ARTIFICIAL INSEMINATION, 8., Cracow, 1976. Proceedings p.69-72.
• ³⁰
  - OLDHAM, C.M.; ADAMS, N.R.; GHERARDI, P.B.; LINDSAY, D.R.; MACKINTOSH, J.B. The influence of level of feed intake on sperm-producing capacity of testicular tissue in the ram. Australian Journal of Agricultural Research, v.29, n.1, p.173-9, 1978.
• ³¹
  - PHADKE, A.G.; MacKINNON, K.J.; DOSSETOR, J.B. Male fertility in uremia: restoration by renal allografts. Canadian Medical Association. M.A. Journal, v.102, n.6, p.607-8, 1970.
• ³²
  - ROCHA, A.; CARPENA M.; TRIPLETT, B.; NEUENDORFF, D.A.; FORREST, D.W.; RANDEL, R.D. Testicular and epididymal function during the peripuberal period in Brahman bulls receiving various amounts of protein degradable in the rumen. Theriogenology, v.45, n.2, p.477-88, 1996.
• ³³
  - ROCHA, A.; CARPENA M.; TRIPLETT, B.; FORREST, D.W.; RANDEL, R.D. Effect of ruminally undegradable protein from fish meal on growth and reproduction of peripuberal Brahman bulls. Journal of Animal Science, v.73, n.4, p.947-53, 1995.
• ³⁴
  - RODRIGUEZ-RIGAU, L.J.; STEINBERGER, E. The Testis and spermatogenesis. In: ZANEVELD, L.J.D.; CHATTERTON, R.T. (Eds.). Biochemistry of mammalian reproduction New York : John e Wiley, 1982. Chap. 1, p.1-35.
• ³⁵
  - ROLLER, M.H.; RIEDEMANN, G.S.; ROMKEMA, G.E.; SWANSON, R.N. Ovine blood chemistry values measured during ammonia toxicosis. American Journal of Veterinary Research, v.43, n.6, p.1068-71, 1982.
• ³⁶
  - SAS INSTITUTE INC. Sas user’s guide: statistics. 5.ed. CARY : SAS INST., 1985.
• ³⁷
  - SMITH, M.L.; LEE, R.; SHEPPARD, S.J.; FARISS, B.L. Reference ovine serum chemistry values. American Journal Veterinary Research, v.39, n.2, p.321-2, 1978.
• ³⁸
  - THOMPSON, L.H.; GOODE, L.; MYERS, R.M.; LINNERUD, A.C. Effect of urea on semen quality and fertility of ruminant males. Journal of Animal Science, v.35, n.1, p.195, 1972.
• ³⁹
  - TSUKAGUCHI, H.; SHAVAKUL, C.; BERGER, U.V.; TOKUI, T.; BROWN, D.; HEDIGER, M.A. Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis. Journal of Clinical Investigation, v.99, n.7, p.1506-15, 1997.

^*

Financiamento: FAPESP Nº 95/2902-1.

¹

Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da USP – SP

²

Departamento de Nutrição e Produção Animal da Faculdade de Medicina Veterinária e Zootecnia da USP – SP

³

Tecnopec Consultoria e Representação Ltda.

⁴

Universidade Paulista, ICS – SP

Publication Dates

• Publication in this collection
  22 Aug 2001
• Date of issue
  Dec 2000

History

• Received
  22 Mar 2000
• Accepted
  30 Nov 2000