GRANT.paper.proposalEDITED

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Yama OE, Gbotolorun SC, Dosumu OO & Kusemiju TO

PRE-CONCEPTION GENDER SELECTION IN WISTAR RATSBACKGROUNDGender determination or sex selection is any techniue or inter!ention

intended to increase the li"elihood o# the conce$tion, %estation and birth

o# a child o# one sex rather than the other 'uman (ertili)ation andEmbryolo%y *uthority document on Sex Selection, +11-. The ability toconsciously select the %ender o# an unborn child is the utmost desire o# most cou$le. This is ex$ected to reduce unto/ard tension and li"elihoodo# do/nsi)in% #amily as a conseuence o# an unendin% attem$t at %ettin%a $articular sex Sex selection and (amily 0alancin% +12 and 3ert) and(letcher, 1454-.

* critical understandin% o# the intricate inter$lay o# mechanisms in the$hysiolo%ical6anatomy o# the #emale re$roducti!e cycles in!ol!ed /ould%o a lon% /ay in brin%in% about balancin% the %ender in a #amily. Thereare sundry %ender selection theories /hich are barely su$$orted by

scienti7c e!idence but are ho/e!er /idely belie!ed to ha!e enjoyed$re!ious successes /ith selectin% their o/n baby8s %ender. 9ost till datestands as mere #ol"loric assertions, :old /i!es; tales or myths and mayactually raise medical uestions. * brie# re!ie/ o# some these methodsand belie!es documented in literature /ould be discussed.


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dee$ rear6entry :do%%y6style;- or shallo/ :missionary style;- $enetration/ould also lead to a $re#erred sex. Other #actors hi%hli%hted by this methodto alter the !a%inal $' include #emale or%asm, !a%ina douchin% Carson,1455- or usin% some dietary techniues re#erred to as :electric #oods; Terra,+4-. 'eat has been sho/n to "ill both ty$es o# s$erm, but mostly the less

$rotected, smaller Y6chromosome hence loose or ti%ht under$ants can aectsex o# a baby Shettles et al., +J and (ertility #riend, +1-. The calori7c!alue o# mothers diet has also bein% $ostulated to aect %ender dierence. Those /hose calorie inta"e is hi%h about +,+ calories- at the time o# conce$tion ha!e a J $er cent chance o# ha!in% a boy /hile those /ith lo/ero# around 1,H calories only ha!e a F $er cent chance o# ha!in% a boyeremy, +5-.Besearch has sho/n that the o!ule controls its electrical char%e in anirre%ular but alternatin% $redictable $attern so as to attract either 6bearin%chromosomes at one time or Y6bearin% chromosomes at another Carter,1444 and 0aby0yGender, +1-. This $henomenon, called the Cyclic

!ariation o# o!ule $olarity, is 7xed #or each s$ecies and s$eci7c to eachone. These inter!als corres$ond to an eecti!e $olarity o# the o!ule areinters$ersed /ith neutral $eriods o# !ariable duration durin% /hich s$eci7cdietary in$ut had a$$eared to ha!e a si%ni7cant in?uence on the sex ratio0aby0yGender, +1-.Other $o$ular $ractices in cou$les attem$tin% at in?uencin% the %ender o#

the unborn child are the :e!en and odd number myth; and the Chinese

%ender chart *shley, +1+-. =n the #ormer, usin% basal body tem$erature as

a $redicti!e %uide to recordin% date o# o!ulation, its !alue is added to the

date o# 7rst day o# L9< $lus the a%e o# the mother. Odd number means a

male %ender /hile e!en number #emale. The Chinese %ender chart reuiresthe Chinese a%e on certain date to be calculated #ollo/ed by Chinese lunar

calendar con!ersion and 7nally $re%nancy and baby %ender chart usin%

Chinese lunar calendar *shley, +1+-.

=n this $resent study all !ie/s /ill be critically examined and the !aryin%

#actor care#ully selected and considered in the ex$erimental animals. The

study /ould be tailored at $ro!idin% e!idences su$$ortin% or re#utin% these

claims.

STATEMENT OF THE PROBLEM

=n nearly all %o!ernments and reli%ious settin%s, contrace$tion and choosin%the sex o# a baby by %ender method usin% natural means #alls into the same

acce$tance ethics. The conseuence o# the increased $re!alence o# :/ron%

%ender; in cou$les is $resently a source o# emotional #riction 3ert) and

(letcher, 1454-. The reliability o# the !arious %ender selection theories in

$redictin% the sex o# a baby has al/ays been a source o# concern to both

#ertility ex$erts and $atients ali"e. S$eci7cally, o!er its s$ectra o#


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assum$tions based on mere anecdotal e!idences2 the s/ollen #ailure rates

are $robably hin%ed on the $aucity o# /ell documented scienti7c e!idences.

* #e/ studies ha!e examined other a%ents on the %ender outcome most o#

them albeit ambi%uous. The o$$osin% !ie/s o# its success ha!e there#ore

not been subjected to objecti!e scienti7c $roo#s.

The use o# mani$ulati!e method to determine %ender /ould alle!iate the

$li%ht o# many anxious cou$le li!in% in the de!elo$in% /orld /here (

treatments are inadeuate and !ery ex$ensi!e. =t is /ith this in mind that the

animal studies to in?uence %ender in estrous rats are im$erati!e.

OBJECTIVES

The o!erall aim o# the study is to $re6determine the %ender o# dams in

:cou$led albino; 3istar rat. The s$eci7c objecti!es are to:

• Serially mani$ulate the !a%inal $' milieu.• =n!esti%ate the eects o# %enetic and some $hysical #actorsN a%e, blood

%rou$, tem$erature and $ressure chan%es as /ell as the date timin%- o# #ertili)ation in?uence on the sex o# a resultin% os$rin%.

• Determine cyclic !ariation o# o!ule $olarity and the immunohistochemicalmar"ers on the )ona $ellucida in !arious estrous $hases.

• Determine the correlation bet/een the %ender o# a dam and the maternaloxidati!e status.

• Se$arate the and Y carryin% s$ermato)oa.

SIGNIFICANCEuality o# li#e ar%uments ha!e been ad!anced as reasons #or sex selection.

Sex choice is said to enhance uality o# li#e more #or a child o# the /anted

sex than a child o# the un/anted %ender. =t is belie!ed to $ro!ide better

uality o# li#e #or the #amily that has the balance it desires. =t $ro!ides a

better uality o# li#e #or the mother, because she /ill under%o #e/er births to

ha!e the desired number o# children o# each sex 3ert) and (letcher, 1454-.


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To address this %a$, /ith an understandin% o# the re$roducti!e $hysiolo%y o#

#emale re$roducti!e cycles an attem$t /ould be made to serially mani$ulate

re$roducti!e milieu to achie!e desired %ender results /ith less cost.

This study in 3istar rat, the 7rst to the best o# our "no/led%e, /ill be o#

immense bene7t as it /ill identi#y in the $' le!els, electrical o!ule

rhythmicity, oxidati!e status as /ell as %enetic and $hysical #actors that are

best reuired to achie!e a desired sex in rat s$ecies. Besults are ex$ected to

thro/ more li%ht on understandin% o# the $atterns o# sex determination.

METHODS

Material a!" #et$%":A!i#al

Six hundred and thirty J- J61F /ee"s *lbino rats Rattus novergicusN

3istar strain- o# both sexes #emales and +4 males-, bred and

maintained at the *nimal 'ouse o# the De$artment o# *natomy, Colle%e o#

9edicine o# the Ini!ersity o# La%os- /ill be used. The rats are to be #ed rat

cho/ and /ater ad libitum, exce$t on days $recedin% serum assays /hen

they /ill only be allo/ed access to /ater 1+ hours. *ll rats /ill be housed in

$lastic ca%es + ratsMca%e- ha!in% dimensions o# x + x 1 cm and so#t

/ood sha!in%s em$loyed as beddin% in the ca%es. They /ill be maintainedunder standardi)ed animal house conditions tem$eratureN +561OC2 li%htN

a$$roximately 1+ h natural li%ht $er day2 humidityN 6A- and

ex$erimentally !aried as $er $rotocol. The animals /ill be allo/ed + /ee"s o#

acclimati)ation to laboratory conditions be#ore the commencement o# the

ex$eriments.

Ge!eral &r%t%'%l Throu%hout the ex$erimental durations, routine ca%e6side examinations /illbe conducted daily to determine the /ellbein% o# the rats sali!ation,rhinorrhea, lacrimation, che/in% ja/ mo!ement, $tosis, suintin%, /rithin%,

con!ulsions, tremors, /ea"ness, lethar%y, re#usal o# #eeds, /ei%ht loss,yello/in% o# #ur, loss o# hair-, stress erection o# #ur, !ocali)ation andexo$hthalmia-, beha!ioral abnormalities, and moribund or dead rats.(ood and /ater inta"e /ill be determined by measurin% the daily le#to!ers inthe ca%es Batnasooriya et al., +-.*ll $rocedures in!ol!in% animals in this study to con#orm to the %uidin%$rinci$les #or research in!ol!in% animals as recommended by the Declarationo# 'elsin"i and the Guidin%


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*merican a%inal $' mani$ulation J

GH6G4 *%e, o!ule $olarity & immunohistochemistryFM%rou$-

1

G16

G1

0lood ty$eN Ty$e*2 Ty$e92 Ty$e*9 and Ty$eO

1M%rou$-

F

G1F6

G1H

Timin% intromission F

G156

G14

9aternal oxidati!e stress +

G@( @ormal #emale G@(- #or matin% G+6G+ 1N1- FTwo hundred and ninety fve (!5) "ale rats

G+6G+

Scrotal tem$erature !ariations F

G+F S$erm sortin% 1G@9 @ormal re%ular males G@9- #or matin% G16GH,

G16G14 1N1-

+F

@OTEN G1 to GH, G16G14 P+F #emale rats that reuire matin%. GH &

G5P 4 rats /ould not be mated.


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Pre&arati%! %+ ,i%'$e#i'al tet rea)e!t a!" a)i!al &H ariati%!

Graded $' solutions used #or !a%inal douchin% /ould be $re$ared in the

De$artment o# 0iochemistry, (aculty o# 0asic 9edical Sciences, Colle%e o#

9edicine, o# the Ini!ersity o# La%os. Douchin% /ith test a%ents /ill be done

usin% rubber $i$ette. >a%inal $' /ould be measured immediately usin% a

standard $' meter and stri$s- be#ore matin%. Each rat /ill be mated at

dierent times durin% the #emale8s rece$ti!ity $eriod and as $er $rotocol.

Sex ratios $ercenta%e male and #emale os$rin%- /ill be recorded at

$arturition.


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/ill be determined based on the $resence or absence o# leu"ocytes, corni7ed

e$ithelial, and nucleated e$ithelial cells accordin% to (elicio, et al., 145F.

3hen the #emale is in $roestrus, mostly nucleated and some corni7ede$ithelial cells are $resent. Some leu"ocytes may be $resent i# the #emale isin early $roestrus. *s the sta%e o# the cycle ad!ances to estrus, mostly

corni7ed e$ithelial cells are $resent. =# the cycle is not interru$ted by$re%nancy, $seudo$re%nancy, or other $henomena, metestrus /ill be%in.9etestrus is a brie# sta%e /hen the cor$ora lutea #orm but #ail to #ullyluteini)e due to a lac" o# $ro%esterone 0yers et al., +1+-. The uterine linin%/ill be%in to slou%h and e!idence o# this is seen in the #orm o# corni7edei$ithelial cells and $olymor$honuclear leu"ocytes $resent in !a%inal s/abs.Some nucleated e$ithelia cells /ill also be $resent in late metestrus. Diestrusis the lon%est o# the sta%es lastin% more than + days. >a%inal s/abs durin%diestrus sho/ $rimarily $olymor$honuclear leu"ocytes and a #e/ e$ithelialcells durin% late diestrus. Leu"ocytes remain the $redominant cell ty$eha!in% remo!ed cellular debris. The cycle then re$eats.

The !a%inal smear o# rats /ill be examined daily #or 1 days, and thoseexhibitin% at least three consecuti!e F6day re%ular estrous cycles *mah et

al., +11- /ill be included in the study.

E/&eri#e!tall. #a!i&*late" a)i!al treat#e!t re)i#e!

Sixty J- sorted #emale rats /ould be randomly allotted into G1 to GJ

treatment %rou$s. Each %rou$ com$rises 1 rats as #ollo/sN

• G1 rats /ill be %i!en orally al"ali)in% K'CO- salts or *l"aline #oods-2

• G+ rats /ill be treated orally /ith *cidic salts @'FCl- salt or *cidic #oods-2

• G rats treated /ith @eutral salts @aCl- or @eutral #oods-2

• GF rats *cidic $' %rou$2 $' solution ?uctuations #rom 1.6J.-2• G rats *l"aline $' %rou$2 $' solution !ariations #rom 5.61F.-,

• GJ rats @eutral $' solution %rou$2 $' H.-.

A)e e0e't1 &%laritie 2e!er). '$ar)e3 eti#ati%! a!"

i##*!%$it%'$e#itr. %+ %*le #e#,ra!e

This cate%ory com$rises #emale rats at onset o# $uberty randomly distributed

in %rou$s GH to G4 /ith each %rou$ com$risin% F rats. Grou$sN

• GH rats in these a%e brac"ets J61F /ee"s old /ould be mated and theeect o# a%e on %ender ratio /ould be determined.

•

G5 rats o!ule $olarity /ill be monitored in the estrous cycle #rom J

th

/ee"6till meno$auseMor u$ to 1 cycles. The contents o# the o!iducts o# euthani)ed rats /ill be ?ushed into a $etri dish to loo" #or oocytes- ase!idence that o!ulation had occurred. Isin% a laboratory char%e analy)erthe $olarities o# the o!um membrane in the estrous cycles /ould bemonitored 9icrosco$y Besource Centre. *natomy o# the ?uorescent9icrosco$e- consecuti!ely at onset o# $uberty until meno$ause in the rats.* de7nite $attern o# $olarity /ill there#ore be established.


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!a%ina. * tender $ressure on either side o# the urethra in the male rat /ill

certainly ex$ose the $enis.

E0e't %+ ar.i!) Te#&erat*re at '%!ta!t &re*re %! t$e )e!"er The %rou$s G+ to G+ com$rise randomi)ed male rats 1M%rou$- /hosetesticular tem$eratures at constant $ressure /ould be altered $rior to matin%

/ith cyclin% #emales. 9ale rats; baseline body tem$eratures /ould 7rst beestimated and external scrotal tem$eratures !aried #rom W+ to W5OC o# baseline rat body tem$eratures. These %rou$s o# rats /ould be mated /ithnormal #emale G@(- in ratio 1N1. Tem$erature !ariation /ith %enderdierence /ill be obser!ed.Se&arati!) t$e &er#at%6%a ,eari!) 7 a!" 8 '$r%#%%#e'ar!ested caudal epididymal s$erm #rom ten 1- euthani)ed male rats in

%rou$ G+F /ould be se$arated into and Y by centri#u%al countercurrent

distribution in a sensiti!e char%ed aueous t/o6$hase system a method

described by 9ario et al., +.

Oral t%/i'it. t*"ie %+ tet a)e!t

LD2 acute and chronic studies on all test solutions /ould be carried out.

Data a!al.i

Data /ill be ex$ressed in 9ean X Standard de!iation SD-. *nalysis o#

!ariance *@O>*- /ith Scheer;s $ost6hoc test /ill be used to analy)e the

si%ni7cance o# dierence and a $robability o# p . considered to be

si%ni7cant.

REFERENCE


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*mah C=, Yama OE, Duru (=, Osinubi **, @oronha CC, O"anla/on *O. Eect o#

"omordica charantia on estrous cycle o# S$ra%ue6Da/ley rats. #acifc $ournal

o% "edical &ciences, +112 51-N H6F5.

*merican


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