THE DOG BREEDER S TOOLKIT

Transcription

1 THE DOG BREEDER S TOOLKIT My Update After the 2016 ISCFR Dr Emmanuel FONTAINE

2 Can you believe that fall is already at our doorstep? I can tell you guys, this is definitely NOT something I am looking forward to Sure fall also means Halloween, candies, orange, yellow and purple trees especially here in Canada. Let s be fair I also enjoy those things! But knowing that, right after, winter is coming Brrr When you know that sooner than later your world will turn into a remake of Frozen, you focus on the positive and enjoy every moment as much as you can. And I can tell you one thing: summer was simply awesome. Weather was great in my part of Canada, we had a lot of family fun. And what made it even better, and special, is that I had the chance to attend to a conference which is very dear to my heart: the 2016 ISCFR.

3 For those of you who wonder what ISCFR means, it stands for International Symposium on Canine and Feline Reproduction. Let s call it the Mecca of the repro specialists in veterinary medicine, because this is where we all gather every 4 years. It is the place to be to connect with colleagues from all over the planet and find out what everybody is working on. It is the place we discuss our different ways of working, share our approaches, think of the future of our discipline. I can t emphasize it enough: it is a great experience that helps all of us bring back home a wealth of knowledge and lots of new ideas to implement in our day to day activities. This year again, we all learnt a lot. And some of those studies that were presented will definitely change the way I approach canine reproduction. Moreover, my US colleague Dr Kesler also had the chance to attend to the 2016 SFT, the Society for Theriogenology conference. It takes place every year in the United States and this is where North-American veterinarians who have a special interest in canine repro gather. He was able to get me one of the abstract book that I thoroughly read.

4 So you see, very busy summer in the field of canine reproduction, no doubt about it! Those two events represent together 2 huge abstract books, 6 days of conference, and more than 400 pages of scientific presentations on canine reproduction. There were data presented on superfecundation (=when you use several males for one female), discussion on biotechnologies of reproduction in canines (which has to do with techniques like IVF In Vitro Fertilization, ICSI Intra Cytoplasmic Sperm Injection, semen/embryo/ovarian cortex freezing, ), presentations of the future of genital ultrasounds Obviously in a one-hour webinar, we won t be able to cover it all. We will only be able to offer you a condensed version. So we came up with a Top 10 of what we believe might be of interest for you dog breeders in your day to day activities. Seat tight, relax, and get ready. Let s dive into those new advances in canine repro that are going to change our and your tomorrow.

5 When canine repro specialists gather, I can tell you one thing: they always speak of infertility cases they have to deal with. Certainly because, as you know, those can turn into complex cases, combining male and female factors. Quick reminder: there is one thing we all know for sure and we all agree on this. #1 cause of infertility in canines is mistiming of the day of breeding. That is why progesterone tests are recommended to determine the day of ovulation and therefore determine the best time to breed the bitch. Find out more about timing of ovulation in bitches here : However, what happens when you do a proper timing of ovulation, you use a male dog that is known to be fertile because he had puppies recently, the bitch is perfectly healthy and in optimal body condition and despite all your efforts and all the efforts of your veterinarian, she is still NOT pregnant? That s a question I m asked on a regular basis. Those cases are those complex mysteries we are always trying to crack. And let s be fair, the differential diagnosis list could clearly go on and on.

6 Infectious diseases are always something to consider. I think of one disease in particular named canine brucellosis (my two cents: if you breed dogs, you MUST know about this one, read this blog here: ). I am a big fan of ultrasounds, I think each infertile bitch that is seen in consultation should get one to evaluate the aspect of the ovaries and the uterus. Uterine disorders are a common cause of infertility as well, and for some of them, the only clinical sign is definitely infertility (check our previous webinar on the topic: ) We usually don t look too much into the genetic side of things these days but I can tell you that in humans they do and they find for instance sex hormone receptors defect that help explain some cases of infertility they deal with. Maybe we will get to that point in the future in dogs as well. As you can see, there are many potential causes. However, you know what I often hear or see in the field? Or what I read a lot on Facebook forums?

7 The bitch is infertile: it MUST be a bacterial infection. Which antibiotic is needed here? I definitely don t think that is the right approach to take in those cases. Don t get me wrong, bacterial infections can cause infertility. In my experience however, they usually come with clinical signs: vaginitis that can be detected with vaginal smears or even better endoscopy, vaginal discharge In a world where antibiotic resistance is on everyone s lips, we need to be careful in the way we use those antibiotics. And I can tell you one thing: they are NOT the go to solution when it comes to canine infertility. And that is something that was again highlighted during the 2016 ISCFR.

8 Since 2009, we have been talking a lot about a disease called endometritis, an inflammation of the uterus that can disrupt fertility. Endometritis is not detectable with standard ultrasound techniques. Some studies have already shown that close to 40% of breeding bitches could suffer from this problem. More about canine endometritis here : Endometritis is an inflammation. When we give some thoughts to what is responsible for this inflammation, I m not going to lie the first thing that usually comes to mind is still bacterial infection. The study from Argentina highlighted on the slide above looked into this. They did uterine biopsies on 56 bitches during routine spay and neuter surgery. For each case, they also sampled the uterus to perform bacteriology. They diagnosed endometritis in 33 bitches (which confirms that this problem is definitely not uncommon). However, the part I find interesting is that they were able to grow bacteria from the uterus in only ONE case.

9 Their conclusions: endometritis is common AND more importantly, bacteria appear not to be relevant in endometritis pathogenesis. To make it simple: it does not appear to be a bacterial disorder. Antibiotics are not needed. On a side note, they also discuss the use of a blood marker called CRP (which stands for C-reactive protein) that could help for diagnosis and prognosis of the disease. That means that a simple blood sample could help suspect the problem in infertile bitches.

10 Take-Away #1 : When it comes to infertility in canines, remember that pure inflammation of the uterus with NO bacterial compound is frequent. I guess now that the question which is one every one s lips is the following: Great we know that endometritis is a common cause of infertility. How do we deal with it? The following study from Poland definitely brings some answers.

11 They tried to find a medical way to approach this problem. They did not go for antibiotics, since they also suspected that endometritis was a pure inflammation. So they went for the use of anti-inflammatory drugs. They used a very specific protocol, combined with intra-uterine inseminations with TCI (=Trans Cervical Insemination, see here ). The treated group consisted of 33 females with I quote a «history of 3 recent unsuccessful consecutive attempts to achieve pregnancy». In those 33 bitches, 10 (43%) of them got pregnant and mean litter size in this group was 5.6±3.7 puppies. I find those results extremely encouraging. Sure we haven t found yet the solution to all fertility issues in the bitch. Those two studies however confirm that endometritis is a frequent problem that impacts fertility, and that new medical approaches can be successful.

12 And most important to me is our take-away #2 : antibiotics are NOT the go-to solution in infertile bitches. That of utmost importance these days. In human medicine, it has been recently identified that there is a uterine microbiome (=bacteria leaving in the uterus and playing an active part in maintaining a healthy uterine environment), the same goes for the normal vaginal bacteria flora in the bitch. Unappropriate use of antibiotics can disrupt this microbiome and in fact create issues that were not there before. Again, don t get me wrong, antibiotics are great drugs we have. Those studies just remind us that we should use them wisely, especially these days with all the known problems related to antibioresistance.

13 The most exciting part of this congress was definitely what was shared in the field of canine neonatology. Remember, neonatology is that period that goes from birth to weaning and we ve touched on it during previous webinars. I think each time we discuss this topic we mention that there are still lots of grey areas and that we still have much to learn on canine neonates. The 2016 ISCFR definitely brought up lots of great information that, I m sure, will definitely change the way we approach neonatology in the field.

14 Most of this ground-breaking work was carried out by a veterinary research group called NeoCare, based at the French veterinary school of Toulouse. I definitely encourage you guys to check their Facebook Page, they are a very active group that share great info in the field of canine reproduction

15 The NeoCare studies are quite impressive for me because they started in 2010 and since then, they have monitored 726 puppies and analyzed more than 15,674 clinical parameters. One thing I can tell you for sure: those numbers are huge in a field like canine neonatology and I m not aware of anything similar so far that has been accomplished. Ground-breaking is definitely not usurped here, believe me! Every piece of information we get to know in this field is important since, in puppies, we are still struggling in average with a 20% neonatal mortality rate, which is far from being negligible. So first, we know that neonatal survival is affected by what we call maternal factors. Those have to do with what happens during gestation, parturition, even lactation and that can have an impact on the newborns. Again, we won t be able to cover everything that was discussed during those 2 congresses but here are some interesting highlights I think you guys should know about.

16 The picture above is what we call a gangrenous mastitis. This purple area you see here in fact tells you that there is a huge abscess developing underneath the skin. This is definitely the most severe form of mastitis that you can encounter in bitches and it will always lead to a rupture of the abscess, leaving a huge hole at this location. Very impressive clearly. I wish that to nobody.

17 This next picture is a more classic form of mastitis, something we would define in veterinary medicine as acute mastitis. Definitely not something uncommon in breeding kennels. If you palpate the affected mammary gland, you will see that it is warm, painful and hard. The color of the milk might be affected, it is usually from brown to red in some occasions. That reminds you why it is so important to palpate the mammary glands of your lactating bitch on a daily basis to make sure that they are not developing mastitis.

18 However, what about this last picture? When we give a quick look at the mammary glands here, everything seems totally normal. I can even tell you that if you were to palpate them as well, you probably would not feel any modifications in terms of consistency. And yet, this is a picture of a bitch also suffering from mastitis Obviously, it is a different kind of mastitis we are dealing with here. This is what we would refer to as a sub-clinical mastitis and I can tell you that this kind of problem often makes us scratch our heads in veterinary clinics. The definition tells it all. There are no real clinical signs to tell us that something is wrong. We would usually suspect it when we see clinical signs on the puppies! They cry, they lose weight they even die sometimes. Definitely a tricky disorder to deal with! Tricky, and not well studied in canines. Which is definitely a problem since, as we just mentioned, it can impact the newborn puppies health. That is also a concern because so far, mastitis in the bitch has only been described as a bacterial infection. Antibiotics are required as part of the medical treatment. If their use is totally justified in the presence of evident clinical signs, well, how can we be sure in the case of sub-clinical ones?

19 Imagine how great it would be if, tomorrow, we could come up with an easy-toperform test that could tell us whether or not there is a sub-clinical mastitis. We could use it before hand, in terms of prevention to make sure puppies nurse at no risk. And worst case scenario, if we end up seeing clinical signs, we could use it as a diagnostic tool to confirm whether or not we are indeed dealing with a subclinical disorder that we need to treat. How great would that be? Well, let me show you something.

20 On the picture above, you can see what we call neutrophils. They belong to a group of cells we call polymorphonuclear cells, and those are considered as markers of inflammation. In other species, ruminants especially, they are used to determine whether there is a mammary inflammation or not. Especially the sub-clinical ones we just mentioned. There are many different ways to count them but one very simple approach consists collecting some milk from the mammary gland, dropping it on a slide and having a look at it under the microscope to perform what we call a cytology. We would evaluate how many of those cells we see. And based on their amount, we would be able to suspect or not- an underlying disease. So why don t we use them in canine reproduction? Again, there have been very very few studies on this topic so far. And the few I remember reading when I prepared my board exam some years ago were saying that their number was highly variable and that therefore it was not predictive at all. I used to tell that to my veterinary students.

21 Things changed after the 2016 ISCFR. The study above is from our friends at NeoCare. They collected 422 milk samples from 50 bitches in order to evaluate an easy-to-use method of inflammatory cell count in the bitch as we just described it. They were able to find a threshold defining mastitis in the bitch. In itself, this is great news because it opens the door for this easy-to-use test on our bitches, and could complement the measures that are already taken to optimize mammary gland health during lactation. They also found out that the prevalence of the disease was 10.7%. That s definitely not negligible in breeding dog population. What is more interesting to me also is that they found out that ANY mammary gland could be affected. We used to say that the last abdominal mammary glands are more predisposed to develop this kind of problem, but in fact, this appears not to be the case. All mammary glands should be evaluated individually if we go down this path then.

22 They also found that the presence of a sub-clinical mastitis was associated with neonatal mortality, but only between D3 and D7 after parturition.

23 That s our take-away #3: subclinical mastitis does pose a risk for newborn puppies health. It reminds us that it is important to maintain those daily measures to monitor mammary glands health during lactation (daily palpation of each gland, checking the color of the milk). Those new diagnostic tools are still under development for sure but I find it is a great first step. Dog breeders could benefit from this research very soon to better detect those sub-clinical and yet potentially problematic disorders.

24 Some of you might say : but if it is a bacterial disorder, why don t we just perform a bacteriology on a milk sample and then treat accordingly? That could sound like a reasonable idea but remember: each mammary glands could potentially be affected. If you go down this path, you would need to check them all. Independently.

25 The conclusions of this study from Italy will also help us a great deal here. They had a rather similar idea than the previous study we just discussed: find a way to detect sub-clinical mastitis. They did things a little bit differently: they collected milk samples and to evaluate presence of inflammation, they did bacteriology AND cytology on their samples. What they found is that isolation of bacteria from milk is very common and is not indicative of mammary gland infection.

26 Take-away #4: Cytology appears to be necessary to correctly interpret bacteriological findings. Therefore, you would need to correlate both to determine if there is a sub-clinical mastitis or not. In my opinion, we have here a really simple tool that can definitely help us optimize mammary gland health during lactation for sure. No doubt dogs breeders will benefit from this in their day-to-day activity.

27 Infectious diseases are a threat we all fear, especially when we have puppies. And in terms of canine infectious diseases, I know there is one that is (and should) be on every dog breeder s lips : canine parvo. In case you believe this is a disease from the past, let me tell you : it is NOT and it remains the most common cause of pediatric mortality in puppies, especially at the time of weaning.

28 Let s imagine a hypothetical situation: - In your kennel, all your bitches are properly vaccinated against parvo (remember, this is still the best way to protect our dogs against it). - You use the right cleaning & disinfecting protocol. You are on top of things on sanitation measures and foot traffic in your kennel. - You have a huge wall surrounding your kennel preventing any life form to get close. - You do not go out and you do not allow anyone in. Ok, I agree, pretty hard to achieve, but let say you can do it. What if I tell you that, despite that, you could still end up with parvo infection in your puppies?

29 Scary, right? However, that is a reality. And I believe you need to know about it. This is what they discussed in the abstract above. They wanted to determine whether vaccinated dams could excrete CPV2 from mating to the end of lactation and so be a potential source of infection in breeding kennels. I ll skip you the details: the answer is YES. Bitches were found to have high viral loads in their feces during lactation. And the later they were in their lactation, the higher the viral excretion! In this study none of those bitches develop any clinical signs of parvo (certainly because they were properly vaccinated) but the fact is that they were actively spreading the disease, clearly indicating that vaccinated dams may contribute to CPV2 circulation in kennels. On a positive note, they also followed the puppies of these dams. And at a certain point, 76% of those puppies were also shedding the virus. The good news is however that only 14% of those puppies experienced diarrhea, most of them remained asymptomatic. And the overall mortality in those puppies was only 3%. Those puppies might have certainly been protected by the immunity they received at birth via the colostrum of their mom.

30 Take-away #5: Parvo MUST always be considered as a risk in breeding kennels, especially for puppies at weaning. Previous studies have already demonstrated that parvo circulation does not always cause the severe hemorrhagic diarrhea syndrome we all fear, but can still cause mild diarrhea in puppies. It is therefore always important to stay on top of things when it comes to sanitary prevention of the disease: sanitation procedures with the right disinfectant of the maternity / nursery will be of the utmost importance, even if you do not allow anybody from outside in your kennel during this period. So in your dog breeder s toolkit you definitely need whatever is required to keep a good sanitation protocol against parvo. Some good info on this here:

31 There is a story I like to tell when I lecture on canine neonatology. When I was a young veterinarian, on a Dec 24th evening while I was doing emergency calls, a couple brought me a 3-day old puppy in consultation. They thought it was not doing well, but were not really sure. I hate to this, but early in my career, I really had no idea how to approach this type of situation. I had been essentially trained in dealing with adult dogs or cats, or growing puppies and kittens. Newborns, that was definitely an unknown territory... Fortunately I learnt a lot since then, and I already shared some of my thoughts on this topic in previous webinars we did that you can check here However, I believe there is still so much to learn in how we handle and assess newborn puppies. I always tell breeders : in your maternity, it is imperative that you have access to a thermometer and a scale. Those are some of the best tools we have to monitor puppies health (see here for more info: ). Thanks to the Neocare studies, I can tell you I learnt many things more at the 2016 ISCFR.

32 First important thing I learnt had to do with neonatal mortality in newborn puppies. The NeoCare studies indeed showed the following : 81.1% of newborn puppies dying during the first 48h of life had a low birth weight. The weight at birth has a huge impact and helps us therefore quickly identify puppies that are more at-risk. That definitely emphasizes the importance of always keeping a scale handy when you have puppies. There is however a trick here: Define low birth weight please.

33 I m sure we will all agree that many factors will influence the weight at birth: the litter size, the nutrition during gestation, the breed... There are average values that are published in certain veterinary textbooks as you can see on the slide above. In the NeoCare studies, they defined it as the «lowest 25% registered values» for a given litter. And you guys can also use your experience here and define low birth weight as weight below what you usually see. One important fact remain in my opinion: it is definitely important to identify those individuals right after they are born. And immediately take extra-care, like for instance milk replacer supplementation. The sooner you ll start taking care of them the better the outcome, that s a certainty! On a side-note, be aware that our friends at NeoCare currently work on growth curves in puppies. Their objective is to be able one day to provide growth curve data per breed. So if you have some of those data (great habit to have is to record your newborn puppies weight on a daily basis), don t hesitate to share with them! The more growth curves we have available, the better it is for the dog breeding community. Just reach out to them via their Facebook Page (and tell them I sent you there ).

34 So identifying puppies with low birth weight is of the utmost importance, but I am sure at this point, many of you might think that this is not necessarily a huge breakthrough in canine neonatology. We now just have more numbers to back up what we already thought. It emphasizes the importance of having a scale at home to weigh your puppies on a daily basis, that s for sure. However, that is a recommendation that has been given for many years already Wait There is more to it!

35 The NeoCare studies introduced a new indicator: Early Growth Rate. I m sure all of you have heard puppies can lose up to 10% of their birth weight during the first day after birth, but then should gain weight. This is written in many textbooks. I myself said that in many of my presentations over the years. The NeoCare studies demonstrated that, in fact, this is NOT true. ANYTIME puppies lose weight, there is a risk. That is what the Early Growth Rate (see slide above) help us identify. The NeoCare studies showed that the optimal cut-off value of growth rate during the first 48 hours of life to assess predictive likelihood of mortality was -4%. Think about it: it means that if a puppy born at 100g loses 4g the first day of life, it is at risk! In fact, if this happens, the risk this puppy dies during the neonatal period increases 8 times! NeoCare studies indeed showed that if puppies lose more than 4% of their initial birth weight during the first day of life, their neonatal mortality rate was 38.5%. While puppies above this threshold experienced a neonatal mortality rate of only 5%. Huge difference, right?

36 This re-emphasizes something we always tell: if there is one thing you definitely need to monitor your puppies health, it is a scale. And remember: they should NOT lose weight AT ALL the first day after they are born. In my past presentations on canine neonatology I kept repeating that there are 2 tools every breeders MUST have in their maternity: a thermometer and a scale. Those studies however described something that might be interesting to complete this toolkit. I always talk about the 3-H syndrome, 3-H standing for hypothermia / dehydration / hypoglycemia (see here: ).We always believed this is one of the most common cause of neonatal mortality in puppies. That s why we recommend the scale and the thermometer, to detect earlier signs like weight loss (now you know that you can also focus on low birth weight) or hypothermia. When I attended to those conferences, there was a special focus on the hypoglycemia part. Until now I always considered it was more of a clinical diagnosis in puppies, when you observe weakness, lethargy, eventually seizures Of course, you can take blood samples to analyze the blood glucose concentration

37 in those puppies. But let face it, in the field, in newborn puppies, this is not something done in routine The result of my colleagues from NeoCare showed a relation between blood glucose levels in puppies and neonatal mortality risk. And there two things that were really striking for me here: - #1: Neonatal mortality risk was increased 4-fold if the puppy s blood glucose concentration was found below 0.9g/L 24hours after birth. I spent a lot of time reading textbooks, and that finding will really change the way I approach canine neonatology I can tell you. Because textbooks mention that hypoglycemia is defined in newborns when blood glucose levels drop below 0.4g/L. Quite a gap, isn t it? The NeoCare studies showed us that problems arise earlier than that definitely. Here you would probably say: «Ok great, but that is mainly for veterinarians who can perform and run blood samples inside their clinic. What is the point for us, dog breeders?» In fact, this is where I was really astonished. - #2: In their studies, those measures were performed directly at the kennel. Right next to the puppies. Some of them were even done by the breeders participating to their studies. I couldn t help but asking: how? Did they only work with breeders with medical skills that know how to draw blood (and even that - drawing blood in a newborn especially if it is not doing wellcan be challenging!). In fact absolutely not.

38 This is what they used : a portable glucometer from human medicine, designed for diabetic people. And instead of pricking their finger to collect a blood sample, they prick the puppy s ear. The key point for my friends at NeoCare was that this is something any breeder could do by himself. Simple, easy, efficient. And it helps detect puppies at risk as soon as 24 hours after they are born. In the future I definitely foresee that this kind of tool could become integral part of the dog breeder s toolkit. It will nicely complement the scale and the thermometer and will allow us to be more accurate at detecting at-risk newborn puppies and immediately take preventive measures to optimize their chances of survival.

39 Those studies therefore definitely help us define simple criteria to better and sooner detect at-risk newborn puppies. Low birth weight, early growth rate and blood glucose levels are now something any breeders can use and that will help determine when to initiate preventive measures to optimize the survival chances of any given puppy. That s my take-away #6.

40 This is great, but wait, there is even more! THE biggest breakthrough that was presented during this conference was without any doubt around canine colostrum. Remember, this is the first milk of the mother that will provide the newborn puppies with immunity AND energy. Read more about it here: Colostrum is enriched in what we call immunoglobulins. Those are immune proteins that, after they are drunk by the puppy, will in fact pass the puppies intestinal barrier to create what we refer to as passive transfer of immunity from the mother to her puppies. By drinking the colostrum, puppies will receive immunity against the diseases the mother is vaccinated for (that is why it is so important to vaccinate them against parvo) and the disease the mother encountered during her life.

41 One thing I can tell you: this immunity transfer is all about timing. - Colostrum is only produced during the first 24 hours of lactation. However, the amount of immunoglobulins it contains dramatically decreases VERY quickly. A decline in the immunoglobulin concentration is noted as soon as 8 hours after beginning of lactation, and there is a 50% by 24 hours! That could become a problem in the case of prolonged delivery, exposing puppies to the risk of deficit of passive immune transfer. - We know moreover from previous studies that colostrum in puppies is only absorbed during the first 12-16h after they are born and it starts to dramatically decrease as soon as 4 hours after birth. After that, the immunoglobulins are no more able to go through the intestinal barrier and will therefore not provide them with this immune protection they totally lack right after birth. So you understand that it is kind of a race right, and you want to make sure that your puppies will drink the colostrum as soon as possible after they re born.

42 I have a question for you here. Look at this picture. You know that a bitch has typically 5 pairs of mammary glands. On which pair would you like the puppies to drink first to make sure he gets the colostrum? And do you think there is one pair that will provide better quality colostrum (which means more immunoglobulins, more energy) than the others? What I usually hear is that colostrum quality should be better in the abdominal mammary glands, since they are the ones that are producing more milk. That the assumption I would have also made. However, what the NeoCare studies showed is that in fact colostrum quality vary widely from one mammary gland to the other! In real life, you currently have no way to predict which one will give the best quality colostrum! Variations from one mammary gland to the other in terms of immunoglobulins vary widely: 42±32.1%!!! Main problem we have so far: there is no qualitative way to assess the colostrum quality in bitches in routine. Refractometers that are used in other species like in mares simply don t work in canines.

43 In canines so far we are therefore not able to predict what the real quality of the colostrum of the bitch is with practical and easy-to-use techniques in the field, and in fact on which mammary gland we would like the puppies to nurse first. That makes it pretty tough therefore to make sure that our puppies indeed get the colostrum we so want them to drink! That s Take-away #7!

44 So we know that puppies need to drink the colostrum to be better protected and get this energy booster right after birth. However, there are huge variations in colostrum quality from one mammary gland to the other. Timing can also be an issue as well, especially in prolonged deliveries. How big a deal is it therefore if puppies don t get to drink it? That s what the NeoCare group focused on in their studies and they found something interesting. They did blood samples in newborn puppies, evaluated their immunoglobulin blood concentrations and determined the optimal cut-off value of immunoglobulin concentrations to assess predictive likelihood of mortality in puppies (it was 230mg/dl). When puppies immunoglobulin concentration was above this value, their neonatal mortality rate was 4.9%. When it was below : 44.4%. That s a huge difference right? And they found out that this concerns 18.1% of puppies.

45 18.1% of the puppies are at risk, and nearly half of them will not make it if they do not receive the colostrum. That s Take-away #8.

46 So now you guys must start thinking : «Ok great, this is a problem but I have no way to really determine if the puppies drank the colostrum properly and doing blood samples in newborns to evaluate their immunoglobulin concentration does not seem to be very practical so what could I do? " The NeoCare studies in fact found a very simple way to assess colostrum intake in puppies: weight gain. They found out that there was a high correlation between early weight gain and colostrum intake, and that early weight gain was a simple and easy way to evaluate if the puppy drank the colostrum or not. It comes back to what we were saying before: having a scale in newborn puppies is still of the utmost importance! Ok, so far what did we learn from the NeoCare studies? That there are factors that can be monitored to detect at risk puppies like low birth weight, blood glucose levels & early growth rate very soon during their growth period. And they also identified that failure of colostral transfer could be a risk factor as well. The great thing is that they did not stop here. They really wanted to come up with solutions to help those at-risk puppies.

47 Their first hypothesis was that energy intake right after birth may be one of the factor limiting puppy survival, and that failure of colostrum transfer and therefore negative early growth rate could contribute to that. So what they did is that they evaluated the impact of an early supplementation of puppies with a milk replacer enriched with a glucose polymer called maltodextrin. Maltodextrin is used in formulas for premature human babies because it is absorbed faster at the digestive tract level and when we consider the weight and the stage of development of puppies at birth, they share many similarities with human premies. Supplemented puppies received the enriched milk replacer every 6 hours from 12 to 48h after birth. Their conclusion : the supplemented group had a significantly higher growth rate at 48h (7.2±7.8% vs 2.9±10.7% in the control group). Remember that they came up with several ways to identify puppies at risk for neonatal mortality. They noticed that supplementing with maltodextrin significantly decreased the number of puppies at risk (16.2% vs 28.4% in the control group).

48 Take-away #9: This study showed the interest of systematic energy supplementation through milk replacer administered immediately after birth. Energy supplementation reduces the number of puppies at risk of neonatal mortality, with an early growth rate <-4%. That is a strategy to look into when an at-risk newborn is detected.

49 As we mentioned earlier, colostrum brings for sure energy, but also immunity. As their previous studies demonstrated also, puppies that did not have an adequate level of immunoglobulins after drinking the colostrum were also more at risk for neonatal mortality. So they tried to use hyper-immune plasma (which is the part of the blood that contains antibodies) they collected on routinely vaccinated dogs. They used it in a slightly different way than it was done in previous studies, since puppies were supplemented with plasma at birth and subsequently every 2 days until day 56. They compared it to a control group into which puppies did not receive any kind of supplementation. They found a significant association between weight gain and supplementation. Moreover, supplemented large breed puppies gained more weight than control large breed puppies (1408±217g vs 815±376g). What they also showed is that the digestive microbiome in those supplemented large breed puppies was also different. Clostridium which is usually the poster picture of the bad bacteria - was less represented in supplemented puppies whereas Lactobacillus, which are seen as «healthy» ones, were more represented.

50 This is of particular interest in large breed puppies since they have been demonstrated to be more susceptible to diarrhea during the pre-weaning period, which is obviously associated with lesser weight gain. Their hypothesis was therefore that hyper-immune supplementation affects the digestive and global health of puppies. The story however does not end here

51 So long term hyper-immune plasma supplementation could be beneficial for puppies but this is not always something practical to achieve because over a 56 day period, well, it does represent a lot of blood to draw and prepare in order to administer it to the puppies. So they tried a similar experiment. This time however, they used a hyper-immune egg yolk powder supplementation. So that must sound a bit weird but let me quickly explain. The concept of hyperimmune egg supplementation is not really something new and is based on the use of what we call the chicken IgY. Those are the immunoglobulins we found in chicken, and they accumulate in the egg yolk. To make it super short, you vaccinate the chicken against diseases that could potentially harm the newborn, they produce specific antibodies against this disease that accumulates inside the egg yolk. You recover the egg yolk, turn it into powder and you have a hyper-immune preparation targeting your pathogens of interest newborns can benefit from. This concept has been proposed as an alternative to antibiotics in f

52 arm animals and egg powder administration has been showed to significantly reduce the risk of GI infections in mice, poultry, piglets and calves. So they obtained a hyper-immune egg yolk powder containing specific antibodies against canine parvo and E coli. We already mentioned the importance of canine parvo but it is good to note as well that E coli is considered today as the most common bacterial cause of neonatal mortality in puppies. They added this egg yolk powder to a canine milk replacer and tried its efficacy. The findings were very similar to what they observed with the hyper immune solution: in large breed puppies especially, supplemented puppies gained more weight during the entire neonatal period. As we discuss before, better growth is the reflection of a better health.

53 Take-away #10: Hyperimmune preparations showed beneficial effects on the health of the newborn puppies..

54 That is the statement that came out of the NeoCare studies. And their studies gave birth to this.

55 This is PRO Tech, the new generation of milk replacers. It contains the maltodextrin we touched on, and thanks to the IgY technology it also contains those immunoglobulins against parvo and E coli that were used in the NeoCare studies. After what I just told you about the studies behind this product, you can understand how excited I was when I found out about this product. It has just been launched in Europe in September. We don t have a launch date yet for North America, but I can tell you one thing: it s coming, and I m definitely looking forward to it.

56 So you see, it has been a busy summer, filled with bursts of scientific excitement. We could not tell you all of it obviously because well, you know, we are quite talkative on those topics but we think the points we presented, especially on neonatology, are going to be real breakthroughs in the field of canine repro. Obviously, we don t have solutions yet to everything, whether it is in the field of infertility as well as neonatology. But coming up with new approaches, new strategies, new tools, is always something exciting, I think we will all agree. As you may have noticed, based on what we learn in 2016, I will have to change several parts of the talks I give on canine repro now. That s the beauty of it somehow. We keep learning as we go brought a lot of great stuff for people like us who are interested in this field. I can tell you one thing: I can t wait to see what s next! The future of this discipline seems bright, believe me

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