Effects of preweaning zeranol implants on reproductive function and growth performance were studied in 45 Simmental-Angus bulls. At slaughter, flow cytometry measurements were made on testicular and sperm cells to determine the effects of zeranol on spermatogenesis. There were no differences in weaning or slaughter weights due to implants. Nonimplanted bulls had larger scrota1 circumferences and heavier testicular weights than bulls given one or two implants. The testes of implanted bulls had a lower proportion of developing germ cells relative  to nonimplanted bulls. The DNA in sperm from implanted bulls was structurally less stable (i.e., more susceptible to denaturation) than DNA in sperm from nonimplanted bulls. Preweaning implantation with zeranol negatively affected testicular function and spermatogenesis, but did not affect growth of yearling bulls

Ballachey, B.E.

Miller, H.L.

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South Dakota State UniversityOpen PRAIRIE: Open Public Research Access InstitutionalRepository and Information ExchangeSouth Dakota Beef Report, 1986 Animal Science Reports1986Flow Cytometry Evaluation of Testis and SpermCells and growth Performance of Bulls Implantedwith ZeranolB.E. BallacheySouth Dakota State UniversityH.L. MillerSouth Dakota State UniversityFollow this and additional works at: http://openprairie.sdstate.edu/sd_beefreport_1986Part of the Meat Science CommonsThis Report is brought to you for free and open access by the Animal Science Reports at Open PRAIRIE: Open Public Research Access InstitutionalRepository and Information Exchange. It has been accepted for inclusion in South Dakota Beef Report, 1986 by an authorized administrator of OpenPRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contactmichael.biondo@sdstate.edu.Recommended CitationBallachey, B.E. and Miller, H.L., "Flow Cytometry Evaluation of Testis and Sperm Cells and growth Performance of Bulls Implantedwith Zeranol" (1986). South Dakota Beef Report, 1986. Paper 26.http://openprairie.sdstate.edu/sd_beefreport_1986/26FLUd CYTOlIETRY EVALUATION OF TESTIS AND SPERn BHD GROCJTB PKRFORHANCE OF BUUS 1- WITE ZERA?NX B. E. Ballachey, H. L. Mi l l e r ,  L. K. J o s t  and D. P. Evenson Departments of Chemistry, Biochemistry Sect ion,  and Animal and Range Sciences BEEF REPORT CATTLE 86-25 E f f e c t s  of preweaning zeranol  implants on reproduct ive  func t ion  and growth performance were s tud ied  i n  45 Simmental-Angus b u l l s .  A t  s l augh te r ,  flow cytometry measurements were made on t e s t i c u l a r  and sperm c e l l s  t o  determine t h e  e f f e c t s  of zeranol  on spermatogenesis.  There were no d i f f e r e n c e s  i n  weaning o r  s l a u g h t e r  weights  due t o  implants.  Nonimplanted b u l l s  had l a r g e r  s c r o t a 1  circumferences and heav ie r  t e s t i c u l a r  weights  than b u l l s  given one o r  two implants.  The t e s t e s  of implanted b u l l s  had a lower propor t ion  of developing germ c e l l s  r e l a t i v e  t o  nonimplanted b u l l s .  The DNA i n  sperm from implanted b u l l s  was s t r u c t u r a l l y  l e s s  s t a b l e  ( i . e . ,  more s u s c e p t i b l e  t o  denatura t ion)  than DNA i n  sperm from nonimplanted b u l l s .  Preweaning implanta t ion  wi th  zeranol  nega t ive ly  a f f e c t e d  t e s t i c u l a r  funct ion  and spermatogenesis,  bu t  d id  no t  a f f e c t  growth of y e a r l i n g  b u l l s .  (Key Words: Zeranol,  Bul l s ,  Tes tes ,  Spermatogenesis, Flow Cytometry.) In t roduct ion  Zeranol,  a r e s o r c y l i c  a c i d  lac tone ,  increases  growth i n  s t e e r s ,  and, a l though t h e  mode of a c t i o n  is  not  c l e a r l y  understood, i t  i s  widely used. With renewed i n t e r e s t  i n  feeding i n t a c t  males f o r  increased  product ion e f f i c i e n c y ,  t h e  a b i l i t y  of growth promotants t o  improve g a i n s  i n  b u l l s  becomes an important cons idera t ion .  E f f e c t s  of zeranol  on growth i n  b u l l s  a r e  n o t  w e l l  e s t a b l i s h e d  and much v a r i a t i o n  i n  response has been reported.  Detr imental  e f f e c t s  of preweaning zeranol  implants  on t e s t i c u l a r  s i z e  and weight have been c o n s i s t e n t l y  observed, bu t  spermatogenic func t ion  and semen q u a l i t y  i n  b u l l s  implanted a t  an e a r l y  age have not  been w e l l  s tudied .  Understanding changes i n  spermatogenesis caused by ze rano l  may he lp  t o  e s t a b l i s h  t h e  mode of a c t i o n  and o v e r a l l  va lue  of t h e s e  implants  t o  promote growth i n  b u l l s .  Flow cytometry i s  a r e l a t i v e l y  new technology wi th  tremendous p o t e n t i a l  f o r  c e l l u l a r  analyses.  Ce l l s  i n  suspension may be  l abe led  wi th  a f l u o r e s c e n t  dye s p e c i f i c  f o r  t h e  component of i n t e r e s t  (e.g., DNA) and passed s i n g l e  f i l e  i n  a sample s tream i n t e r s e c t e d  by a l a s e r  beam. Upon l a s e r  e x c i t a t i o n ,  f luorescence  i s  emi t ted  from the  c e l l s .  The f luorescence  i s  de tec ted  by photomul t ip l ie r  tubes,  converted t o  a d i g i t a l  analog s i g n a l  and subsequently analyzed by a computer i n t e r f a c e d  t o  t h e  flow cytometer. Advantages of flow cytometry inc lude  r ap id  measurement of a l a r g e  number of c e l l s ,  simultaneous measurement of s e v e r a l  v a r i a b l e s  pe r  c e l l  and ease  of c l a s s i f y i n g  and counting subpopulat ions of c e l l  types.  Previous s t u d i e s  ( see  t h e  preceding paper i n  t h i s  r epor t )  have demonstrated t h e  va lue  of flow cytometr ic  ana lyses  t o  s tudy male reproduct ive  funct ion  and reproduct ive  toxicology. The purpose of t h i s  research  was t o  u t i l i z e  flow cytometry t o  e v a l u a t e  t e s t i s  and sperm c e l l s  of y e a r l i n g  beef b u l l s  implanted w i t h  ze rano l  p r i o r  t o  weaning. Experimental Procedure C a t t l e .  Forty-f ive 3 / 4  Angus x 1 / 4  Simmental b u l l  ca lves  averaging 30 days of age were weighed and randomly d iv ided  i n t o  t h r e e  t rea tment  groups: control-no implant (011, one ze rano l  implant (11) and two ze rano l  implants  ( 2 % ) .  Calves i n  t h e  t r e a t e d  groups were implanted w i t h  36 mg of ze rano l  under t h e  s k i n  on t h e  lower backs ide  of t h e  e a r  according t o  manufacturer 's  recommendation. Ninety days l a t e r  ca lves  i n  t h e  2% group were given a second ze rano l  implant.  A f t e r  weaning a t  an average age of 205 days, ca lves  were t r anspor t ed  approximately 300 mi les  t o  a f e e d l o t  f a c i l i t y .  B u l l s  were f ed  a grower r a t i o n  c o n s i s t i n g  of a l f a l f a  hay o f f e r e d  ad l i b i t u m  and 5.5 l b  of corn d a i l y  f o r  90 days followed by a f i n i s h i n g  r a t i o n  f o r  155 days u n t i l  s l augh te r .  The f i n i s h i n g  r a t i o n ,  fed  ad l i b i tum,  cons i s t ed  of 84.0% corn, 10.5% corn s i l a g e ,  4.8% soybean meal and .7% minera l  supplement. B u l l s  were s laughtered  a t  an average age of 450 days. F ive  days be fo re  s l a u g h t e r ,  s c r o t a 1  circumferences were measured and, from 10  randomly s e l e c t e d  b u l l s  of each t rea tment  group, an e l e c t r o e j a c u l a t e d  semen sample w a s  c o l l e c t e d  and placed on i c e .  A t  s l augh te r ,  t e s t e s  and v a s  de fe rens  from a l l  b u l l s  were c o l l e c t e d  and placed on i c e  and t e s t i s  weights  were recorded. From b u l l s  which had been e l e c t r o e j a c u l a t e d ,  a t e s t i c u l a r  biopsy was a l s o  taken  f o r  measurement by flow cytometry. Within 6 hours of c o l l e c t i o n ,  vas  sperm were recovered from t h e  vas  deferens  of e l e c t r o e j a c u l a t e d  b u l l s .  C e l l  P repa ra t ion  g& Sta in ing .  Flow cytometry measurements were made on -e j a c u l a t e d  sperm samples t h e  day of c o l l e c t i o n .  I f  necessary ,  samples were d i l u t e d  w i t h  phys io log ica l  s a l i n e  s o l u t i o n  t o  lower t h e  c e l l  concen t r a t ion  p r i o r  t o  measurement. Vas sperm were c o l l e c t e d  by s t r i p p i n g  t h e  con ten t s  of t h e  vas  deferens  i n t o  s a l i n e  s o l u t i o n .  Vas sperm and t e s t i c u l a r  c e l l s  were obta ined  on t h e  same day and t h e r e  were t o o  many samples t o  be run by flow cytometry t h a t  day. Therefore,  v a s  sperm were mixed w i t h  g l y c e r o l  and f rozen  a t  -1000 C u n t i l  f low cytometry measurements were made approximately 2 months l a t e r .  Each t e s t i c u l a r  biopsy,  weighing approximately 1 g, was placed i n  s a l i n e  s o l u t i o n  a t  t h e  t i m e  of c o l l e c t i o n .  Six t o  e i g h t  hours a f t e r  c o l l e c t i o n ,  t h e  biopsy w a s  minced w i t h  s c i s s o r s  t o  form a c e l l u l a r  suspension, f i l t e r e d  f r e e  of t i s s u e  clumps and measured by flow cytometry t o  determine DNA and RNA content  of each c e l l .  T e s t i c u l a r  c e l l s  and e j a c u l a t e d  and vas  sperm were s t a i n e d  w i t h  a c r i d i n e  orange (A01 dye and measured by flow cytometry. The procedure involves  t r e a t i n g  t h e  c e l l s  w i t h  a s o l u t i o n  con ta in ing  de t e rgen t  (al lows dye b e t t e r  access  t o  c e l l  components) and mild ac id ,  then  s t a i n i n g  w i t h  AO. T e s t i c u l a r  c e l l s  con ta in  bo th  DNA (double-stranded) and RNA. When A0 binds w i t h  DNA, i t  f l u o r e s c e s  green, and, when i t  b inds  t o  RNA, i t  f luo resces  red. Measurement of green and red  f luo rescence  a l lows  q u a n t i f i c a t i o n  of t h e  r e l a t i v e  DNA and RNA content  and, on t h i s  b a s i s ,  p ropor t ions  of t e t r a p l o i d ,  d i p l o i d  and haploid c e l l s  i n  a sample can be  determined. The haplo id  c e l l s  a r e  t h e  developing germ c e l l s .  I n  sperm c e l l s ,  t h e  s t a i n i n g  procedure i s  c a l l e d  t h e  sperm chromatin s t r u c t u r e  a s say  (SCSA). RNA i s  no t  p re sen t  i n  sperm and, because of condensat ion of t h e  chromatin (DNA and a s soc i a t ed  p r o t e i n s )  a s soc i a t ed  w i t h  maturat ion,  t h e  a c i d  t rea tment  p o t e n t i a l l y  induces p a r t i a l  dena tu ra t ion  of t h e  DNA s o  t h a t  both double- and single-stranded DNA a r e  present .  A0 dye w i l l  f l u o r e s c e  green when bound t o  double-stranded DNA and r e d  wi th  s ingle-stranded DNA. Thus, r a t i o s  of green t o  red f luorescence  measure the  s u s c e p t i b i l i t y  of DNA t o  dena tu ra t ion  and provide information on t h e  chromatin s t r u c t u r e  of t he  sperm. The amount of dena tu ra t ion  was q u a n t i f i e d  by alpha-t ( a t ) ,  def ined  a s  t h e  r a t i o  of red t o  t o t a l  ( r ed  + green) f luorescence.  The mean (Xat) and c o e f f i c i e n t  of v a r i a t i o n  (CVat) of t h e  a t  d i s t r i b u t i o n  were used t o  desc r ibe  t h e  ex ten t  of denatura t ion .  Previous s t u d i e s  have shown t h a t  h igher  a t  va lues  a r e  a s soc ia t ed  wi th  poor semen q u a l i t y  and decreased f e r t i l i t y .  To measure morphological abnormal i t ies ,  sperm from each sample were s t a i n e d  wi th  Eosin dye and smeared onto g l a s s  s l i d e s .  A t  l e a s t  200 c e l l s  pe r  b u l l  were scored by l i g h t  microscopy t o  e s t ima te  percentage of abnormal sperm heads. Resul t s  and Discussion Growth of Bulls .  Performance da ta ,  t e s t i c u l a r  weights  and s c r o t a l  circumferences a r e  shown i n  t a b l e  1. There were no s i g n i f i c a n t  d i f f e r e n c e s  i n  i n i t i a l ,  weaning o r  s l a u g h t e r  weights among t h e  t h r e e  t reatment  groups. Gain t o  weaning (420, 409 and 434 l b  f o r  01, 11 and 21, r e spec t ive ly )  and f e e d l o t  ga in  (504, 476 and 485 l b  f o r  01, 11 and 21, r e spec t ive ly )  a l s o  d id  not  vary wi th  t reatment .  Bu l l s  i n  t h e  nonimplanted group had heav ie r  t e s t i c u l a r  weights and l a r g e r  s c r o t a l  c ircumferences a t  s l a u g h t e r  than b u l l s  given one o r  two implants.  The implanted groups (11 and 21) d id  not  d i f f e r  i n  t e s t i c u l a r  weight o r  s c r o t a l  circumference. T e s t i c u l a r  Samples. T e s t i c u l a r  haploid,  d i p l o i d  and t e t r a p l o i d  c e l l s  can b e  d i s t ingu i shed  by FCM on t h e  b a s i s  of c e l l u l a r  DNA and RNA content  a f t e r  s t a i n i n g  wi th  AO. A h ighe r  propor t ion  of haploid c e l l s  i s  a s soc ia t ed  wi th  more e f f i c i e n t  spermatogenesis.  Within t h e  haploid c e l l  populat ion,  maturing germ c e l l s  can b e  separa ted  i n t o  propor t ions  of round, e longat ing  and elongated spennat ids  because during maturat ion chromatin s t r u c t u r e  becomes more compact and t h i s  is  as soc ia t ed  wi th  decreased green f luorescence  due t o  r e s t r i c t i o n  of A0 binding. Decreasing red f luorescence  i s  a l s o  noted, r e s u l t i n g  from decreasing RNA content  a s  t h e  spermatids.  mature. Propor t ions  of c e l l  types  i n  t h e  t e s t i c u l a r  b iops i e s ,  determined by computer a n a l y s i s  a f t e r  measuring samples by flow cytometry, a r e  presented  i n  t a b l e  2. B u l l s  i n  e i t h e r  t h e  11 o r  2 1  groups had an increased  propor t ion  of d i p l o i d  c e l l s  and decreased propor t ion  of haploid c e l l s  r e l a t i v e  t o  01  b u l l s  (P<.05) .  Proport ions of spermatid types wi th in  the  haploid c e l l  populat ion d id  not  vary  s i g n i f i c a n t l y  wi th  treatment.  Sperm Samples. Mature sperm, whether obtained from the  vas  deferens o r  an e j acu la t ed  semen sample, a r e  cha rac te r i zed  by a  nucleus with highly condensed chromatin and devoid of RNA. Normal sperm s t a i n e d  wi th  A0 should emit l a r g e l y  green f luorescence  ( r e f l e c t i n g  ds  DNA content )  and minimal red  f luorescence.  I n  sperm wi th  abnormal chromatin, t h e  DNA w i l l  be  more s u s c e p t i b l e  t o  denatura t ion ,  r e s u l t i n g  i n  increased  red f luorescence  and h igher  a t  values.  The Eat and CVat of e j acu la t ed  and vas  sperm samples a r e  l i s t e d  i n  t a b l e  3 .  For both sperm samples the. Xat was h ighes t  from 21 b u l l s .  The CVat f o r  e j a c u l a t e d  sperm samples d i d  not  vary  s i g n i f i c a n t l y  w i t h  t rea tment ,  a l though t h e  0 1  group had t h e  lowest  and t h e  21 group t h e  h ighes t  average va lues .  For vas  sperm, d i f f e r e n c e s  were seen between t rea tments ,  w i t h  0 1  b u l l s  having lower average (=Vat v a l u e s  (18.42) than  t h e  11 (23.49) and 21 (27.14) groups. Percentages of morphologically abnormal sperm measured by l i g h t  microscopy a r e  a l s o  presented  t a b l e  3.  For e j a c u l a t e d  samples, b u l l s  i n  t h e  21 group tended t o  have t h e  h ighes t  average p ropor t ion  of abnormal sperm. Treatment e f f e c t s  were more pronounced f o r  vas  sperm, s i n c e  01  b u l l s  had a lower average p ropor t ion  of abnormals than  implanted b u l l s  (27.6, 40.7 and 41.7% f o r  01, 11 and 21, r e spec t ive ly . )  Although e f f e c t s  of ze rano l  on t e s t i c u l a r  development and spermatogenesis  of b u l l s  were demonstrated between t rea tments ,  cons ide rab le  v a r i a t i o n  among b u l l s  w i t h i n  t r ea tmen t s  was a l s o  evident .  Several  b u l l s  of t h e  0 1  group had h igh  l e v e l s  of abnormal sperm and a t  va lues .  B u l l s  of t h e  21 group were c o n s i s t e n t l y  lower i n  p ropor t ion  of haplo id  c e l l s  and and (=Vat of t h e  vas  sperm than  t h e  average of t h e  0 1  group. More v a r i a b i l i t y  e x i s t e d  among t h e  11 b u l l s ;  some had t e s t i c u l a r  and sperm flow cytometr ic  va lues  i n  t h e  "normalw range ( s i m i l a r  t o  01) and some were i n  t h e  "poor" range ( s i m i l a r  t o  21).  I n  conclusion,  t h i s  r e sea rch  i n v e s t i g a t e d  t h e  e f f e c t s  of preweaning z e r a n o l  implants  on growth and spermatogenesis of b u l l s .  Weaning and s l a u g h t e r  weights  were not  improved by implanta t ion ,  bu t  sperm c e l l s  i n  implanted b u l l s  were adverse ly  a f f e c t e d  by zeranol .  The de t r imen ta l  e f f e c t  of reproduct ive  func t ion  i s  of major concern i f  t h e  b u l l s  a r e  being considered f o r  breeding  purposes,  s i n c e  i t  would l i k e l y  r e s u l t  i n  reduced male f e r t i l i t y .  TABLE 1. MEAN LIVE WEIGHTS, TESTICULAR WEIGHTS AND SCROTAL CIRCUME'ERENCESa Treatment group Measurement Control 1 2 sEM~ I n i t i a l  wtc,  l b  13 0 135 135 9.5 Weaning w t ,  l b  550 5 44 569 24.5 S laughter  w t ,  l b  11 6 8 11 27 11 42 28.4 T e s t i c u l a r  w t ,  g 63 7d 496e 463e 28.8 Sc ro t a1  circumference, cm 37.8d 35 . l e  34.3e .63 a n = 15 p e r  group. SEM = s tandard  e r r o r  of t h e  mean. I n i t i a l  w t  = w t  a t  time of f i r s t  implant (about 30 days of age) .  d*e  Means i n  t h e  same row wi th  d i f f e r e n t  s u p e r s c r i p t s  vary  (PC .019. TABLE 2. MEAN VALUES OF TESTICULAR VARIABLESa Treatment group V a r i a b l e  Cont ro l  1 2 S E M ~  % d i p l o i d  c e l l s  10.7C 15.5d 20. gd 2.61 % t e t r a p l o i d  c e l l s  11.1 14.1 13.2 1.21 % hap lo id  c e l l s  78.2C 70.4d 65 .gd 2.86 a n = 10  p e r  group. SEM = s t a n d a r d  e r r o r  of t h e  means. c s d  Means i n  t h e  same row w i t h  d i f f e r e n t  s u p e r s c r i p t s  v a r y  (P< .05) .  TABLE 3. MEAN VALUES OF SPERM VARIABLES Treatment group Sperm type  V a r i a b l e  Cont ro l  1 2 SEMa - E j  a c u l a t e d b  Xat .31d .31d .36e -02  CV at 16.18 17.50 19.40 2.06 % abnormal 24.6 28.6 41.4 5.42 - at .23d .23d . 26e .01 ma, 1 8 . 4 2 ~  23 .4ge 27.14e 2.21 % abnormal 27.6d 40.7e 41.7e 5.19 a SEM = s t a n d a r d  e r r o r  of t h e  mean. n = 9 f o r  c o n t r o l  group; n = 7 f o r  one and two implan t  groups.  C n = 10  p e r  group. d.e Means i n  t h e  same row w i t h  d i f f e r i n g  s u p e r s c r i p t s  v a r y  (Pc.05). 

Flow Cytometry Evaluation of Testis and Sperm Cells and growth Performance of Bulls Implanted with Zeranol

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Flow Cytometry Evaluation of Testis and Sperm Cells and growth Performance of Bulls Implanted with Zeranol

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