# Females From Mostly Male Litters



## Maren Bell Jones (Jun 7, 2006)

Okay all, I need everyone's help, especially experienced breeders. I am writing a practice grant proposal for my neuroethology class based on the research we've done in my research lab on the intrauterine position effect. Basically, in the mouse, the uterus has two horns (similar but not identical to the dog) and the pups fit in like peas in a pod. If a pup is in between two males, it is denoted 2M. If between two females, it is 2F. If between one of each, it is 1MF. There are numerous behavioral effects, such as female 2M pups tend to more socially aggressive towards conspecifics (both male and female), have extended estrus cycles (2F females tend to have 4-5 day estrus cycles, 2M females are more like 5-8 days), be less likely to mate, and have a longer anal/genital distance (which makes it harder to sex the pups at birth). This is because they get an extra prenatal "dose" of testosterone by their adjacent brothers. 

The focus of my practice grant proposal is to identify if something similiar happens in dogs. In other words, many familiar with dog behavior and physiology have heard of some females getting more socially aggressive after ovariohysterectomy (though not all females do this). I'd like to "see" (keeping in mind I am not going to physically do this research) if this is due to the intrauterine position effect that has already been characterized in numerous rodent species. 

As dogs and cats have a somewhat similar set up to rodents, *has anyone noticed that females from all male or male dominated litters are more socially aggressive?* 

Remember, this isn't a "real" project, but I think it is very very interesting and no formal research has been conducted on dogs, just rodents, to see if it works the same way. AH HA! I smell a PhD/DVM project! :wink:


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## Don Turnipseed (Oct 8, 2006)

That study has already been done Maren, with pretty conclusive results.


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## Maren Bell Jones (Jun 7, 2006)

In that case, please enlighten me, Don.


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## Don Turnipseed (Oct 8, 2006)

I just couldn't help myself Maren. I am pulling your leg but it kept me chuckling while I fixed dinner. Actually, I don't think I have kept records that would give me the insight you are looking for. Thanks


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## Woody Taylor (Mar 28, 2006)

Don Turnipseed said:


> I just couldn't help myself Maren. I am pulling your leg but it kept me chuckling while I fixed dinner. Actually, I don't think I have kept records that would give me the insight you are looking for. Thanks


Okay, I laughed my butt off on that one. HAH. Well done, Don. You are my new hero, that was great.

Maren, he gotcha.


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## Don Turnipseed (Oct 8, 2006)

Maren's googling trying to find that study Woody. LOL


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## Connie Sutherland (Mar 27, 2006)

Wow, what a poo-poo head!

I am laughing, I admit.......


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## Woody Taylor (Mar 28, 2006)

Don Turnipseed said:


> Maren's googling trying to find that study Woody. LOL


It actually hits home for me because my job is to come up with new tech gadgets for my company to make...there's always one smarta$$ in the back of the room saying "Already been done." And they are usually right. From now on, I will call that person "Turnipseed." :lol:


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## Connie Sutherland (Mar 27, 2006)

Woody Taylor said:


> Don Turnipseed said:
> 
> 
> > Maren's googling trying to find that study Woody. LOL
> ...


"Simpsons did it." 

(South Park lament)


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## Don Turnipseed (Oct 8, 2006)

I hope there is a bit of a sense of humor here becuase I notice that Maren described how the 2F's act, the 2M's act....but no mention was made of the 1MF's. I wonder how they act?


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## Maren Bell Jones (Jun 7, 2006)

RAWR! :evil: Do NOT mess with the grad student at the end of the semester!!! We are stressed out enough as it is. See if I give you all free vet advice in a couple years! Who's gonna be pwned then? 8) :lol: :lol: 

Actually, I didn't even bother googling it (or PubMed or Medline searching it, for that matter) cause I had already been doing it all afternoon. :roll: :roll:

Anyone actually wanna weigh in on this one for real though? Please?


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## Kristen Cabe (Mar 27, 2006)

Jessie was the ONLY female in her litter. She's only 6 months old (or will be on the 6th), but I can try to help by answering any of your questions. She's snoring right now from her crate. I don't think I've ever heard her snore before. :lol: :lol: 

Forgive my newbiness, but what exactly are you asking when you ask about social aggression?


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## Maren Bell Jones (Jun 7, 2006)

Thanks for at least trying to answer, Kristen.  Basically, especially for those who have had numerous litters, *in general* do females born in otherwise all male litters tend to be more socially aggressive (i.e.-trends towards being more territorial and suspicious of strangers and strange dogs, more dominant or pushy over known dogs and perhaps people, possibly harder in temperament, etc) than females born in all or mostly female litters?


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## Jeff Oehlsen (Apr 7, 2006)

Yes I have seen this. The latest example is "Kira" one of our clubmembers dogs. She was the only female and terribly socially aggressive. AND right from the start. She was like a week older than some GSD pups I had and we had to take her away from them. TOOOO much.

Just one example.


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## Don Turnipseed (Oct 8, 2006)

I don't recollect any mostly all male litters but several all female litters. I will look through my records and see. I I happen to keep a female put of a predominately male litter I will know how she acted. The problem is, most of my females are brought back to me for stud service because the owners can't find males that are not afraid of them. I am thinking of one female in particular that had a very short breeding life before I quit trying to breed her and she was rough, but I think there were females in that litter also. It will take a while to look through them all but in a day or so I will let you know.


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## Kristen Cabe (Mar 27, 2006)

> socially aggressive (i.e.-trends towards being more territorial and suspicious of strangers and strange dogs, more dominant or pushy over known dogs and perhaps people, possibly harder in temperament, etc


Um, well, this pretty much describes Jessie to a tee. :lol: If she doesn't know you, you can forget trying to pet her because if you try, she's going to tell you off. After she's had a warm-up-to-you period, she's okay, but she isn't a run-up-to-everyone-wanting-attention puppy. She runs to the fence and barks her fool head off at the neighbors, passing dogs, and Jak. She sounds like a full-grown male when she barks - her bark is deeper and more scary than Jak's.  :lol: I've also caught her trying to hump Adam's leg. :lol: On the other hand, though, she will submit very easily without a fight. I can't remember how many males/females were in the litter before Jessie's, but I can ask next time I go to training if you want me to. They are 9 or 10 months old now I think.


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## Lynn Cheffins (Jul 11, 2006)

I got two female puppies from different litters this summer. The one from the female dominated litter(one male puppy) is the really forward and more dominant with other dogs but very willing to please people. The one from the predominantly male litter is a submissive pee-er and more submissive with other dogs but is really darn stubborn. Some of this is just the inherited temerment of the dogs. I think it would be interesting to compare a predominantly male and a predominantly female litter from the same parents.


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## Selena van Leeuwen (Mar 29, 2006)

not messing with you, I thought there *was* an official study on that.Have read it somewhere, but is probably an European study.

What I recall was it like you discribe with the mice. More dominant, male-like females.

Could it be possible it has something to do with the father? Father is the one who is responsible for the gender (like humans), some males do inherit lots more males than females (with different females-female is resposible for how many pups are born). 
If there are more males in the litter, statistically you would have more 2 M´s in the litter.

It is said that if the male is very dominant by nature/genetics, it is likely he gives many males. So very dominant- high testostonelevel- gives many males?

for example: our late Rocky, is father of different litters, very dominant dog by nature. His offspring is about 70/80 % males, in some litters there was no female at all.


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## Greg Long (Mar 27, 2006)

Just because a dog is predisposed to being more socially aggressive,doesnt mean that you will see any or all of this behavior.The environment the pup is raised in will greatly affect the behaviors as will the influence of the dam.I dont see how you can get an accurate conclusion from this unless you either study hundreds of litters or control the environment from birth.You would also have to study females from predominantly all female litters.It is interesting though.


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## Lacey Vessell (Nov 18, 2006)

I would offer up my bitch as an example as she is from a litter of 6 males and 2 females - the other female from the litter died at about 2 weeks old. I personally feel that her temperament has more to do with genetics though - as I knew both the sire and dam very well and both had an edge to them - the sire tolerated people as did the dam although the dam was less forgiving. The breeder kept one of the males from this litter and he is the same, temperament wise, as my bitch.

This breeder has bred the dam of my bitch four times - only once to my bitch's sire (he died shortly after the pups were whelped  ). The male pups outnumbered the females in two of the other litters (7-2, 5-4,) In one litter there was 6-6. These three litters were all sired by another male that he owns, who is what I would consider a very social dog. I would say that only one of the females out of the three other breedings mentioned would be considered socially aggressive - that female is out of the 5-4.


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## melissa mims (Jul 12, 2006)

From _Handbook of Applied Dog Behaviour and Training_ Volume 1, pg 186:

"A possible couse for the variable effect of castration on sexual behaviour and aggression may be due, in part, to the influence of perinatal androgenization. The puppy's nervous system is androgenized just before birth and after birth by a surge of testosterone....These effects [of masculinizing females] have been demonstrated in other species...and are theoretically possible in dogs [sic]...(Overall, 1977). In fact, Coppola (1986, reported in Borchelt and Voith, 1996) has found that female dogs born in litters predominantly composed of males are more likely to exibit dominance aggression and various masculine tendencies as adults."

Coppola, MC(1986). Dominance aggression in dogs. Master's thesis. Department of Psychology, Hunter College, New York, NY

Overall K (1997). _Clinical Behavioural Medicine for Small Animals_. St. Louis: CV Mosby.


My girl was one of three females in a litter of 11. I think she was born between males. Has a will to power, for sure, that is kept in check with calm handling, absolute limits, and bitework.


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## Maren Bell Jones (Jun 7, 2006)

Thanks everyone. 

Greg, yes, experimental design is a lot more tricky with dogs than with mice, but it can be done based on numerous other studies I've read. I will post the experimental design and the rest of the proposal when I am done writing the paper by next week. To actually determine whether or not a female is a 2M, you would need to deliver the pups via c-section a day or two before birth and record their intrauterine position then. So a female doesn't need to be from an all male litter to know it is 2M, you just have to c-section it to see. 

Don, and that was another thing that was striking about 2M female mice versus 2F female mice is that males definitely prefer to mate with 2F females. Maybe the 2M ones are too butch and scary? I dunno. :lol:

Melissa, yes, thanks. It has been mentioned in the literature as a possibility. It's also difficult to find people's masters thesis, especially before they were published electronically like mine will have to be. I was going to check the Borchelt and Voight book at our vet med library this afternoon.


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## Don Turnipseed (Oct 8, 2006)

"Don, and that was another thing that was striking about 2M female mice versus 2F female mice is that males definitely prefer to mate with 2F females. Maybe the 2M ones are too butch and scary? I dunno. "

Maren, I have gone through about 50 litter reg and see no point in going farther. Most litters are close to 50/50 or heavy on the female side. I can't recall ever having an all male litter. The ones that I found that were marginal...I didn't raise the pups.. This makes the above statement questionable. I think most were probably 1MF or 2F's

"It is said that if the male is very dominant by nature/genetics, it is likely he gives many males. So very dominant- high testostonelevel- gives many males?"

I can't buy that Selena. I have produced far more females and all my males are extremely dominate....as in never posture or growl with each other or strange dogs.


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## Jeff Oehlsen (Apr 7, 2006)

Quote: I can't buy that Selena. I have produced far more females and all my males are extremely dominate....as in never posture or growl with each other or strange dogs

I don't get it.


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## Don Turnipseed (Oct 8, 2006)

I see where that could be confusing. I must have had a lapse in the middle of a thought. They don't growl or posture....as in a dog gets aggressive with them, they just silently drop the hammer on them....like a lightning bolt. Rotties and such come charging them throwing saliva every direction showing a high level of aggression, my males just stand there calmly and wait for them to get there....but they never do. They always break off about 10' to 12' away and suddenly nervously find something to smell....at which time my dogs usually walk over and hike their leg on them. Very interesting behavior and kind of a slap in the face. Winchester used to walk up to dogs showing aggression and lean his shoulder into them and hold his eye about three inches in front of theirs and lean on them. They would cease being aggressive and step back and he would step forward until he totally backed them down. Never was a good time to yell at him because he was keyed. According to the quote from Selena there is some theorising that dominate dogs throw more males. Yet they never bother a small dog even if it is very aggressive.


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## Bob Scott (Mar 30, 2006)

I just read this cause I spent a ton of reading time the last few days when the power was off here. Reading by candle light kin cross yer eyes after 5-6 hours.   
I'll try and find it. I do know it was commented on in one of the three volumes of Handbook of APPLIED DOG BEHAVIOR AND TRAINING by Steven R. Lyndsay. 
I'll try and find it.


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## Bob Scott (Mar 30, 2006)

Bob Scott said:


> I just read this cause I spent a ton of reading time the last few days when the power was off here. Reading by candle light kin cross yer eyes after 5-6 hours.
> I'll try and find it. I do know it was commented on in one of the three volumes of Handbook of APPLIED DOG BEHAVIOR AND TRAINING by Steven R. Lyndsay.
> I'll try and find it.


Found it!
They comment that even in mixed litters, '"if the female is between two males in the uterus she appears to be influenced by the presence of vagrant testosterone carried in amniotic fluids, although it is not certain whether such a hypothesized osmotic mechanism is involved".
The reference they mention is
Knol BW and Egberink-Alink ST (1989).
Treatment of problem behaviour in dogs and cats by castration and progestagen admininistration: A review. Vet Q, 11:102-107.


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## Don Turnipseed (Oct 8, 2006)

Be interested in hearing what the author has to say Bob. I seldom read dog books as there is just to much BS in most of them for my taste. Most are a compilation of what they perceive to be happening....and I just don't agree with a lot of it. Then again, there are certain readings that I find fascinating. I will post one on the breeding forum that is excellent.


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## Liz Monty (Oct 22, 2006)

Melissa, I do not know much about breeding, but I was always under the impression that you can tell during birth if the female is in between two males in the uterine horn. I was surpirsed to hear that an ultra sound is the only way to know how they are placed. I believed in the past that when the pups are being delivered - as the first pup presents from one horn, the next pup will present from the opposite horn and the birthing continues in that fashion. I guess, it is possible that when one horn is fuller than the other, then two pups from the same horn could come out one after the other, skipping the opposite horn at some times. Would this be correct in why the ultra sound is necessary for the result to be known??


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## Maren Bell Jones (Jun 7, 2006)

I haven't come across anything in either the dog or the mouse literature on more aggressive animals having more of the dog or mouse equivalent of XY spermatozoa as I do more with hormones than with genetics. I might look into it. Though I do recall that in certain rural communities that are environmentally exposed to a good mix of pesticides that they tend to have significantly more girl babies than boys. The Y chromosome doesn't have a ton of stuff on it besides the sex determining gene SRY and things (surprise surprise) important to male sexual dimorphism, like the production of sperm. Though they work in concert, I don't _think_ social aggression/dominance (not necessarily the same thing) of the sire and sex determination are directly linked. From an evolutionary perspective, I don't think it makes sense (whether by genetics and/or hormones) for more socially dominant male animals to produce more male sperm as eventually, you are going to have all your male descendants all fighting each other with almost no females to mate with. 

BTW, for those who are curious, 1MF animals are behaviorally and physiologically in the middle between 2F and 2M animals. But not literally! I'll have to dig up the figures from my advisor's many papers on this (he came up with this in the early 80s but now is one of the world leaders in environmental endocrine disruption research...it's cool having a very prolific advisor!), but here's a very simplified version:


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## Liz Monty (Oct 22, 2006)

Oops, in the above post I meant Maren, not Melissa, sorry.


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## Maren Bell Jones (Jun 7, 2006)

> Melissa, I do not know much about breeding, but I was always under the impression that you can tell during birth if the female is in between two males in the uterine horn. I was surpirsed to hear that an ultra sound is the only way to know how they are placed. I believed in the past that when the pups are being delivered - as the first pup presents from one horn, the next pup will present from the opposite horn and the birthing continues in that fashion. I guess, it is possible that when one horn is fuller than the other, then two pups from the same horn could come out one after the other, skipping the opposite horn at some times. Would this be correct in why the ultra sound is necessary for the result to be known??


Liz, the way we determine the intrauterine position in mice is that we c-section them on gestation day 18 (they have a 19 day gestation period) and we lay the pups out on a paper towel in the same way they were arranged. When you can have 10-15+ in a litter, no way am I going to wait around for that sort of thing. Plus in mice, there is generally more pups in the right horn than the left for some reason, so I am not sure how they "take turns" exactly. The rodent uterus is a little different than the carnivore uterus. Rodents have a duplex uterus with two separate horns and two separate cervices leading to one vaginal opening while dogs, cats, etc have a bipartite uterus with two mostly separate horns and one cervix. Humans have a simplex uterus with no horns. So the order of parturition may or may not work the same, but in an experimental design, you could ultrasound the pups, but they'd have to be delivered via c-section to be absolutely sure of the position. 

The funny thing is, like the source Bob cited and like a few others I read this afternoon, everyone says "it appears that it happens" or "it seems that it would happen," but it doesn't seem like anyone's done the darn thing in the way I'll be proposing. In research articles, it's always good to say what they'd like to see done next. This type of experiment is said, "we should do this!" but they still haven't. :roll: If it works in rodents and pigs, it likely would do the same in dogs and cats, but this hypothetical experiment I propose would show it (hopefully!). :lol:


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## Bob Scott (Mar 30, 2006)

Quote:
Liz, the way we determine the intrauterine position in mice is that we c-section them on gestation day 18 (they have a 19 day gestation period) and we lay the pups out on a paper towel in the same way they were arranged. When you can have 10-15+ in a litter, no way am I going to wait around for that sort of thing. Plus in mice, there is generally more pups in the right horn than the left for some reason, so I am not sure how they "take turns" exactly. The rodent uterus is a little different than the carnivore uterus. Rodents have a duplex uterus with two separate horns and two separate cervices leading to one vaginal opening while dogs, cats, etc have a bipartite uterus with two mostly separate horns and one cervix. Humans have a simplex uterus with no horns. So the order of parturition may or may not work the same, but in an experimental design, you could ultrasound the pups, but they'd have to be delivered via c-section to be absolutely sure of the position. 

Then there's the good ol possum. Now that's a REAL interesting bit of anatomy in the male.


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## Liz Monty (Oct 22, 2006)

Ok, I understand much better now why you are following the method this way, me and biology, genetics have not worked well together :lol: :lol:


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## Maren Bell Jones (Jun 7, 2006)

Yup, all marsupials except the big kangaroos have this...errr...interesting feature.


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## Don Turnipseed (Oct 8, 2006)

Maren, I noticed that the males are blue in the illustration you put up which makes me wonder, and maybe you know why. At the time of birth, the female pups seem stronger than the males. This is apparently true also in cattle. I will generally lose more male pups than female. Could it be that they are between two females and don't get "enough" testosterone. This is a legitamate question. There is normally a bigger loss of bull calfs also as I understand, which would have nothing to do with who is between what.


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## Maren Bell Jones (Jun 7, 2006)

When c-sectioning the mouse pups, it is usually the smaller pups (either male or female) who are less vigorous, which makes sense. Sometimes they can be fine if fostered, but often not if below a gram in weight. It is probably more due to intrauterine growth restriction (i.e.-blood flow off the uterine arteries that the pups get from the placenta varies depending where the pup is) than testosterone level. However, if they are intrauterine growth restricted and they do in fact live, the mouse pup as an adult can become very large to the point of being obese because they can go through a ballistic growth rate right after weaning to catch up to its brothers and sisters. Cows don't typically have "litters" unless they are freemartins, so I don't know why male calves have a harder time of it. Then again, I know very little about cattle other than I would prefer not to include them if I end up being a mixed practice vet. Being simultaneously pooped on, peed on, and kicked while doing a pregnancy check with your arm up their butt is not my idea of a good time. :lol:


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## Daryl Ehret (Apr 4, 2006)

> has anyone noticed that females from all male or male dominated litters are more socially aggressive?


I have a couple females that are very dominant, and keep them separated all times. Both are from roughly 50/50 male/female litters though. Ema's very male-like, lifts her leg to pee (has a hairy back-side...Jeff's kind of dog :wink: ). She sometimes instigates confrontation with other females, assertively but not agressively if that makes any sense. Both need to be the "alpha" and neither will back down from the other, but both are very tolerable of young 'uns agressive behaviors. They come from 3:2 and 2:3 litter ratios of male to female. I've notice some largely male litters lately, from the Jinopo kennel I think, but for the most part I think Don's right; most are 50/50 or leaning toward more females. Some male-specific traits are inherited from the dam-side too, so that might be taken into account when speaking of "complex" or "multifactoral" characteristics. Genetic defects underlying low sperm count are possibly even inherited through the dam's side.


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## Daryl Ehret (Apr 4, 2006)

> ...Although a mother's role in sex selection is well-documented, little attention has been paid to the father's role in this phenomenon....
> ...until now, most researchers have focused on the mother's role in skewing sex ratios...
> ...in some species, the healthier the mother, the more likely she is to produce sons...
> ...Among European deer, males with high-quality sperm are more likely to sire sons than daughters...
> ..."Sons inherit the fertility of their fathers,"...


ScienceNOW Daily News
30 November 2006
http://sciencenow.sciencemag.org/cgi/content/full/2006/1130/5


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## Maren Bell Jones (Jun 7, 2006)

For anyone who is interested, here is my final draft of the paper. I didn't include quite all of it just because a section or two gets repetitive. Remember, this a hypothetical experiment and project, though the professor thought the actual concept and experiment was very interesting. Then again, he works on crawfish, so eh... 

Introduction:

Though precise statistics are difficult to determine, millions of animals die in American animal shelters each year. While some are turned over through no fault of their own, behavior problems, such as dominance aggression as perceived by the owner, are commonly cited reasons for the dog to be relinquished. Many additional dogs are turned over to veterinarians by their owners to be euthanized for similar reasons. Furthermore, dog bites and attacks due to aggression account for an average of twelve to twenty fatalities in the United States annually. The many thousands of reported bites (though there are likely many more unreported) cost homeowners insurance companies hundreds of millions of dollars in filed claims.1 In addition, children are the most common victims of fatal and serious dog bites.2 

More studies on the physiological basis of dog dominance and social aggression are needed. One area of interest focuses on examining possible reasons why some dogs from a litter of presumably similar genetics seem to be more predisposed towards aggression than their littermates, even to the point they face euthanasia because they are overly socially aggressive to humans and other animals. The effect of prenatal hormone exposure due to intrauterine position has been a well-characterized model in rodents that may offer a possible explanation as to why some dogs are naturally more aggressive than their siblings even before the onset of sexual maturity. By investigating this possibility, it could shed more light onto the phenomenon of canine aggression and possibly prevent more attacks and fatalities in the future. 

Background:

The intrauterine position effect was first characterized in rodents and later in pigs.3-5 Mice and other rodents have a duplex uterus, which includes two completely separate uterine horns and cervices leading to a single vaginal canal. Dogs and other members of the order Carnivora have a bipartite uterus with two mostly separate horns and one cervix. However, both species are litter-bearing mammals with similar gestational arrangements in which the developing pups are positioned like peas in a pod. In mice and other rodents, fetuses developing adjacent to one another share the blood supply coming from the uterine arteries and veins. While sexual differentiation is occurring during the final days of gestation, the release of the sex hormone testosterone by male fetuses and estradiol by the females also affects those adjacent to the fetus. As shown in Figure 1, a fetus in between two males is designated 2M, between two females is 2F, and between one of each is 1MF. 










Figure 1: This schematic demonstrates how the developing fetuses are arranged in the mouse duplex uterus. The dog bipartite uterus is very similar except there is one cervix instead of two separate cervices. In the figure, the male pup second down from the right ovary is in between two other males and would be designated 2M, the pup third down is between a male and a female (1MF), and the pup fifth down is in between two females (2F). (Adapted from Even 1994)

Although the fetal blood hormone levels on the day before parturition in a 2M versus a 2F male or female differ in the levels of only picograms per milliliter, the behavioral and physiological differences between the two are profound. 1MF pups are considered middle of the road between 2M and 2F pups both behaviorally and physiologically. For example, in terms of reproductive differences, 2F females come into first estrus sooner, have shorter anogenital distances, and their estrus cycles are of a shorter duration (a mean of 4.5 days in 2F females versus a mean of 6.5 days in 2M females). 2M males also have heavier seminal vesicles than 2F males. In terms of behavior, additional differences emerge. Males choose 2F females in estrus over 2M females in estrus approximately 80% of the time.4 2M females also show an age-dependent increase in aggressive behaviors such as dominance mounting of other females.6

Because of the similarities in the arrangement of the dog uterus during gestation to the already well-described mouse uterus, using the dog as a model should be a straightforward way to determine the effect of the intrauterine position by utilizing several known behavioral and physiology assays.

Experimental Design:

Female German shepherd dogs at least two years old from similar lines will be mated with unrelated stud male German shepherds during estrus. Pregnant dams will be fed the same diet appropriate for pregnant and lactating bitches throughout gestation and lactation. Diet will also be controlled for the pups from weaning to adulthood. On gestational day 60, pups will be delivered via caesarian section and sex ratios recorded to establish the intrauterine position for each pup. Blood samples of each pup will be collected as well as a blood sample from the dam. The levels of 17 -estradiol and testosterone will be measured in the blood serum via radioimmunoassay. All pups will wear a color-coded collar until they are old enough to be implanted with a microchip for identification purposes.

During the period of birth to eight weeks of age, the pups will be handled on a daily basis for maintenance, tracking growth, observation and vaccinations by veterinary staff, and for socialization purposes. At eight weeks of age, the pups will be moved to separate indoor/outdoor kennel settings with continued daily human interaction and exercise as adults. Starting at six months of age and repeating every six months until 24 months of age, the dogs will be tested for aggression in the following manner. The following stimuli will approach within one meter of their kennel enclosure to evaluate territoriality and in a novel kennel enclosure out of their territory as previously described8:

1)	a strange adult human male
2)	a strange adult human female
3)	a strange adult dog of the opposite sex being walked on lead by a familiar human (if subject is a male, an adult female in estrus will be walked by)
4)	a strange adult dog of the same sex (if subject is a female, a proven adult stud male will be walked by)

All reactions to the stimulus will be videotaped for approximately two minutes to incorporate time before the introduction of the stimulus and after the departure approximately 60 seconds later. Analysis of the videotape will include investigation for signs of aggression (i.e.-body posture, facial expressions, and vocalizations characteristic of dominant/aggressive versus submissive/avoidance behaviors) at the various stages of development. Following each testing, a blood sample will be taken for serum estradiol and testosterone levels.

When the dogs are fully mature at 24 months old, they will be evaluated for shyness, avoidance, defensiveness, and their reaction to novel auditory, visual, and tactile stimuli by following the temperament test protocol developed by the American Temperament Test Society, Inc. As of 2005, nearly 2800 German shepherd dogs have taken the test with an 83.1% pass rate, so the breed has been well-characterized for these behavioral assays.9

Outcome Assessment:

Using both the fetal and post natal sex hormone serum levels and the behavioral assay results based on the intrauterine position, we can statistically analyze whether or not the dog also follows the model previously established in various rodent species and pigs. If it seems like the serum levels of estradiol and testosterone do not correlate with the arrangement of the pups in utero, further study into the transport system of hormones between the fetus, the dam, and other fetuses via the placenta would be useful. If the behavioral assays selected for the proposed experiment do not yield significant findings, more sensitive anatomical or physiological might be more appropriate until the behavior by each subset of the intrauterine groups can be more fully assessed. As the dog is a novel model system for the intrauterine position effect, any significant data from these experiments would be very helpful in establishing and characterizing of the effects of late prenatal endogenous hormones.

Future Directions:

After establishing the behavioral effects for the model of the intrauterine position on the dog, the next step would be to evaluate how gonadectomy before or after sexual maturity of the dog and bitch further affects the adult phenotype with respect to the intrauterine masculinization or feminization of the pups. While numerous studies have suggested that while castration is effective in ameliorating some behavioral issues in male dogs, performing ovariohysterectomies on some bitches might actually exacerbate certain pre-existing behavior problems.10-12 Since only a subset of females exhibit a post-spay increase in aggression, this has been speculated in the literature to be related to masculinazation due to intrauterine position.13 However, no formal studies to date have been performed.14 

The vast majority of dogs obtained as puppies from animal shelters are spayed or neutered after adoption. In addition, most veterinarians typically recommend gonadectomy to their clients before the dog reaches sexual maturity around 6-12 months of age if it is not of breeding quality. As spay and neuter surgeries are very routinely performed on pet dogs to prevent or to lessen behavior problems as well as preventing unwanted litters, investigation of how hormone exposure based on intrauterine position can affect behavior in spite of (or perhaps in addition to) gonadectomy is critical to our understanding of aggression. 

Insofar as clinical practicality to veterinarians and applied animal behaviorists, some research has suggested that hormone replacement therapy for spayed females exhibiting aggression following ovariohysterectomy might be helpful in addition to behavior modification.10 If a bitch was suspected to be an 2M individual either because it was in an otherwise all male litter or from ultrasound or caesarian section and the client wished to spay the animal, the addition of a hormone replacement therapy regime could prevent heightened post-spay aggression. This in turn would lessen subsequent behavior problems between the dog and humans or other dogs leading to possible relinquishment by the owner or euthanasia. In conclusion, many genetic and environmental factors comprise what we label as social aggression in canines. The effects of hormones on late prenatal development are yet another relatively unexplored component of this behavioral phenomenon. Targeted research into this area could lead to greater understanding and possible prevention of dog bites and the euthanasia of more animals through pharmacological means and behavioral modification.

References:

1) Dog Bite Law. 2006. http://www.dogbitelaw.com

2) Weiss H, Friedman D, Coben J. Incidence of Dog Bite Injuries Treated in Emergency Departments. J Am Med Assoc 1998; 279:51-53. 

3) vom Saal F, Bronson E. Sexual Characteristics of Adult Female Mice are Correlated With Their Blood Testosterone Levels During Prenatal Development. Science 1980; 208:597-599.

4) vom Saal F. Sexual Differentiation in Litter-Bearing Mammals: Influence of Sex and Adjacent Fetuses in utero. J Anim Sci 1989; 67:1824-1840.

5) Ryan B, Vandenbergh J. Intrauterine Position Effects. Neurosci Behav Rev 2002; 26:665-678.

6) vom Saal F, Finch C. Reproductive Senescence. In: Knobil E, Neill J, eds. Physiology of Reproduction. 2nd ed. New York: Raven, 1988; 2351-2413.

7) Even MD, Laughlin MH, Krause GF, vom Saal FS 1994. Differences in blood flow to uterine segments and placentae in relation to sex, intrauterine location, and side in pregnant rats. J Reprod Fertil 1994; 102: 245252.

8 ) Kim H, Yeon S, Houpt K, Lee H, Chang H, Lee H. Acoustic Feature of Barks of Ovariohysterectomized and Intact German Shepherd Bitches. J Vet Med Sci 2005. 67:281-285.

9) American Temperament Test Society, Inc. 2005. http://www.atts.org/statistics.html

10) OFarrel V, Peachey E. Behavioral Effects of Ovariohysterectomy on Bitches. J Small Anim Pract 1990; 31:595-598.

11) Neilson J, Eckstein R, Hart B. Effects of castration on problem behaviors in male dogs with reference to age and duration of behavior. J Am Vet Med Assoc 1997; 211:180-182.

12) Spain C, Scarlett J, Houpt K. Long-term Risks and Benefits of Early-Age Gonadectomy in Dogs. J Am Vet Med Assoc 2004; 224:380-387.

13) Overall KL. Sex and Aggression. Canine Practice 1995; 20:16-18.

14) Borchelt P, Voith V. Aggressive Behavior in Dogs. Readings in Companion Animal Behavior 1996; 217-239.


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## Bob Scott (Mar 30, 2006)

Very interesting! I'm not any kind of statician but how many litters would you have to have in order for the experiment to be considered valid?


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## Maren Bell Jones (Jun 7, 2006)

I've got a sticker by my desk that says "We don't need no stinkin' statistics!" That should give you an idea of my feelings on the matter. As long as the n is high and p value is low, that's all I know. 8) Seriously though, we like to have between 25 and 50 litters when we do experiments like this in mice. May or may not be possible with dogs due to numerous controls on using dogs for scientific research. However, they are not being harmed in any way nor would they need to be euthanized at the end of the experiment, so the numbers wouldn't be bad.


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## Bob Scott (Mar 30, 2006)

Could you get any of the larger kennels to aid you in it or would that loose to much control over it?


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## Maren Bell Jones (Jun 7, 2006)

As long as they were maintained under pretty much identical situations with equal amounts of socialization, same housing, diet, similar lines, it would probably be fine. That's kind of the difficult thing about behavioral studies is that everything must be pretty much identical.


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## Bob Scott (Mar 30, 2006)

Makes sense and I doubt most kennels are going to change their routines for the sake of a science expeiment.


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## Woody Taylor (Mar 28, 2006)

Maren Bell Jones said:


> I've got a sticker by my desk that says "We don't need no stinkin' statistics!" That should give you an idea of my feelings on the matter. As long as the n is high and p value is low, that's all I know. 8) Seriously though, we like to have between 25 and 50 litters when we do experiments like this in mice. May or may not be possible with dogs due to numerous controls on using dogs for scientific research. However, they are not being harmed in any way nor would they need to be euthanized at the end of the experiment, so the numbers wouldn't be bad.


Regarding sample size...what's your metric of interest? What are you gonna measure that's ultimately telling you what you want to know?


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## Don Turnipseed (Oct 8, 2006)

I fail to see what the point od this study is. Of course I fail to see the point of having an ultasound done on a bitch to see how many pups there are....which is wrong as much as it is right, ot to see how many of what is in the litter. Knowing isn't going to change a thing, you are still going to get what you get. With this study, is there a point? Or is it a matter of just knowing. They are still going to be what they are regardless.


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## Maren Bell Jones (Jun 7, 2006)

Woody, the endpoints to compare to one another would be:

-fetal estradiol and testosterone levels in their blood serum measured by radioimmunoassay

-a scoring system of aggression and territoriality probably using similar methods as the Kim et al paper I cited

-the pass fail rate of the temperament test

Don, the basic experiment is more of a discovery based experiment than an a hypothesis driven based experiment. Discovery is pretty much what it sounds like: you figure out a possible model and then see if it works. Hypothesis based says you know your model (well...in theory) and now let's tweak it to show what happens and how it deviates from the original model. So yes, you're going to get what you're going to get, but it is interesting to the body of science to say WHY you are going to get what you'll get. The vast vast majority of studies in science aren't going to make it into _JAMA, JAVMA, Nature_, or _Science_, but they help add to the collective knowledge of WHY (very important to a scientist!).

However, to tell the gods of grants to give you money, you have to show them why it is of interest. So even though this is a hypothetical experiment and it is highly interesting on its own, the dog bite problem etc is the best way to invoke a why other than just because the experiment is interesting. Not everything in discovery based science is going to cure cancer, AIDS, and so on, so most of it just is an addition to the body of knowledge kind of thing. Problem is you can't just say that on a grant proposal. Darn... :wink: Anyways, there was a study that suggested that doing hormone replacement therapy to spayed bitches might alleviate this post spay increase of aggression, so this would be the groundwork to why that phenomenon happens in the first place.

Edit: Woody, let me be the first to say that your avatar scares me.


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## Don Turnipseed (Oct 8, 2006)

I can see it in a few years Maren. "Whoa dude, that is a 2m bitch... way cooool!" :lol:


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## Maren Bell Jones (Jun 7, 2006)

Yes, Don, so we will now know the perfect recipe for a good hard as nails female: get a 2M puppy, spay her, and watch the carnage. :twisted:


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